Effects of ryanodine on cardiac contraction, excitation-contraction coupling and “treppe” in the conscious dog

J Mol Cell Cardiol 27, 2111.-2121 (1995)

Effects of Ryanodine on Cardiac Contraction, Excitation-Contraction Coupling and "Treppe" in the Conscious Dog Bernd Kalthof, Naoki Sato, Mitsunori Iwase, You-Tang Shen, Israel Mirsky, Thomas A. Patrick and Stephen F. Vatner Department of Medicine, Harvard Medical School, Brigham & Women's Hospital, Boston, and the New England Regional Primate Research Center, Southborough, MA O1772, USA (Received 11 October 1994, acceptedin revisedfornI 28 March 1995) B. KALTHOF, N. SATO, M. IWASE, Y.-T. SHEN, I. MIRSKY, T, A. PATRICK AND S. E VATNER.Effects of

Ryanodineon

Cardiac Contraction, Excitation-Contraction Coupling and "Treppe" in the Conscious Dog. Journal of Molelcular and Cellular Cardiology (1995) 27, 2111-2121. The effects of ryanodine on left ventricular (LV) function and hemodynamics were studied in 16 conscious dogs, chronicaUy instrumented for measurements of LV pressures and dimensions. Systemic infusion of ryanodine (0.5--4/2g/kg i.v.) resulted in a dose-dependent depression of cardiac contraction. For example, ryanodine, 4 pg/kg i.v., decreased LV fractional shortening by 30.5 +_4.1%, LV dP/dt by 41.5 +4.0% and Vcfc by 37.8 _+4.1%, while increasing the isovolumic relaxation time constant, tan, from 23.1 _+1.4 to 34.1 _+3.6ms without a major effect on preload or aftedoad. Ryanodine also depressed (P
Introduction

ryanodine binding results in SR Ca2+ depletion by induction of a long-lasting open subconducting state of the SR Ca 2+ release channels (Rousseau et al., 1987), whereas at higher concentrations, a blocking effect can be achieved (Meissner, 1986). Due to its ability to specifically decrease SR Ca2+ content, ryanodine has been widely used for in

The natural plant alkaloid, ryanodine (Jenden and Fairhurst, 1969) binds specifically to the calcium (Ca 2+) release channel of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle (Meissner, 1986). At concentrations below IO0/~M,

Please address all correspondence to: Stephen E Vatner, New England Regional Primate Research Center, One Pine Hill Drive, P.O. Box 9102, Southborough, MA 01772-9102, USA. 0022-2828/95/i02111 + 12 $12.00/0

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vitro studies of excitation-contraction coupling in a variety of cardiac muscle preparations (Banijamali et al., 1991; Bose et al., 1988: Ezzaher et al., 1992: Fabiato, 1985: Furnival et al., 1970: Hadju and Leonard, 1961; Hunter et al., 1983; Lewartowski et al., 1990; Sutko and Willerson, 1980). In agreement with its SR Ca 2÷ depleting action the reported in vitro effects of ryanodine include a decrease in cardiac contractility, intracellular Ca 2÷ transients and SR Ca 2÷ content. The effects of ryanodine in vivo are less well characterized. A number of studies were conducted with ryanodine in anesthetized and in conscious, uninstrumented animals more than 30 years ago (Kahn et al., 1964; Procita, 1958), before the mechanism of ryanodine action was clarified. These early in vivo studies were limited to the investigation of basic hemodynamic effects in anesthetized animals and to toxicity evaluation in conscious, uninstrumented animals. Both approaches were characterized by the use of high, often lethal doses of ryanodine. More recently in vivo studies were focused on the investigation of potential antiarrhythmic effects of ryanodine against digitalisinduced arrhythmias in guinea-pigs (Zakharov et al., 1991) and ischemia-induced ventricular fibrillation in conscious dogs (Lappi and Billman, 1993). Lew (Lew, 1993) investigated the effect of ryanodine on the volume-induced increase in LV contractility in anesthetized dogs. None of these studies, however, investigated the effects of ryanodine on excitation-contraction coupling in vivo, which was the main objective of the present investigation. The first goal of this investigation was to examine the dose-related effects of ryanodine on left ventricular (LV) function and systemic hemodynamics in conscious, chronically instrumented dogs. A second goal was to examine the actions of ryanodine on standard protocols for the investigation of cardiac excitation-contraction coupling, e.g. mechanical restitution and postextrasystolic potentiation. Finally, we investigated the effect of ryanodine on the "Bowditch" or "Treppe" effect (Bowditch, 1871), the heart rate dependent positive staircase of cardiac contraction. The overall goal was to gain a better understanding of excitation-contraction coupling in the conscious animal, and furthermore, to potentially reconcile the controversy regarding the relative importance of the "Treppe" in conscious (I-Iiggins et al., 1973; Noble et al., 1969) v more isolated or open-chest preparations (Boerth, et al., 1969; Dale, 1932; Lendrum et al., 1960; Monroe and French, 1961).

Materials and Methods Instrumentation Adult mongrel dogs 01 = 16), weighing between 23 and 35kg, were sedated with xylazine (0.3 mg/ kg, i.m.), and anesthetized with thiamylal sodium (10-15 mg/kg, i.v.). Following tracheal intubation and ventilation with a respirator (Harvard Apparatus, South Natick, MA, USA), halothane anesthesia (1.0-2.0 vol% in oxygen) was maintained during surgery. With the use of sterile technique and through an incision in the fifth left intracostal space, a solid state miniature pressure transducer with a frequency response of 1 ld-Ig (Konigsberg Instruments, Pasadena, CA, USA) was implanted in the LV cavity through the apex for high fidelity measurements of LV pressure. Tygon catheters were implanted in the descending thoracic aorta and left atrium to measure aortic and left atrial pressures. Piezoelectric ultrasonic dimension crystals were implanted in all dogs on opposing anterior and posterior endocardial surfaces of the left ventricle to measure LV internal diameter and on opposing endocardial and epicardial surfaces in the same equatorial plane to measure wall thickness. The endocardial wall thickness crystal was implanted obliquely to avoid damage to the myocardium between the two wall thickness crystals. Pacing leads were placed on the right ventricular wall and the left atrium. The pericardium was left open and the thoracotomy was closed. After surgery the animals were allowed to recover for 2-3 weeks before beginning experiments. The dogs used in this study were maintained in accordance with the Guide for the care and use of laboratory animals of the Institute of Laboratory Animal Resources, National Council (Department of Health and Human Services publication No (NIH) 85-23, revised 1985) and the studies were approved by the standing committee on animal care of Harvard Medical School.

Experimental measurements Statham strain gauge manometers (model P23 ID, Statham Instruments, Oxnard, CA, USA) connected to the chronically implanted catheters were calibrated with a mercury manometer and used to measure aortic and left atrial pressures. LV pressure was measured with the solid state miniature pressure gauge calibrated in vitro with a mercury manometer and in vivo using left atrial and aortic catheters and the strain gauge manometers. An

Effects of Ryanodine on Cardiac Contraction ultrasonic transit time dimension gauge was used for measurements of LV internal diameter and wall thickness. Briefly, the dimension gauge demonstrates a voltage linearly proportional to the transit time of ultrasonic impulses travelling at a velocity of 1.58 × IO" mm/s between 5 MHz crystals. The position of all catheters and crystals was confirmed at autopsy. The experiments were conducted in conscious, unsedated dogs resting comfortably on their right side. In order to minimize reflex effects and also to depress cardiac function, in five dogs ganglionic and muscarinic blockade was induced by systemic infusion of hexamethonium bromide (30mg/kg, i.v.) and atropine (0.1 mg/kg, i.v.). A stock solution of 5 mg/ml ryanodine (Calbiochem, La Jolla, CA, USA) was prepared in distilled water and stored in the freezer at - 2 0 ° C . Solutions for i.v. administration were freshly prepared at the day of the experiment by dilution of the stock solution with 0.9% NaCl solution as necessary. Dose-response effects were examined in 12 conscious dogs with the following protocol for i.v. infusion of cumulative doses of 0.5, 1, 2 and 4#g/kg ryanodine: infusion with 0.1 #g/kg/min ryanodine for 5 rain (total: O. 5 pg/kg), additional infusion with 0.1 #g/kg/min for 5 min (cumulative total: 1 #g/ kg), additional infusion with 0.25 #g/kg/min for 4 min (cumulative total: 2 #g/kg), additional infusion with 0.5 #g/kg/min for 4 min (cumulative total: 4#g/kg). Each infusion step was followed by a 10 min period prior to recording to allow stabilization of the ryanodine effects. For other experiments ryanodine was infused with 0.25 #g/kg/min for 16 rain to achieve a dose of 4 #/kg, also followed by a 10 min stabilization period. No significant differences in the effects of ryanodine at 4 #g/kg were noted for the two different infusion protocols. Mechanical restitution and post-extrasystolic potentiation of LV contraction were determined in seven conscious dogs before and after infusion of ryanodine 4 #g/ kg. To study this, the heart was paced at a constant rate (150 beats/min, e.g. 400 ms pulse intervals) with right ventricular pacing until a steady-state had been reached (up to 3 min). Two extrastimuli were then introduced after the cessation of steadystate pacing, also with right ventricular pacing. The interval between the end of steady-state pacing and the first extrapulse was increased in 50 ms increments from 2 5 0 m s to 9 0 0 m s for the determination of mechanical restitution, while the interval between the first and the second extrapulse was fixed at 600 ms for the determination of postextrasystolic potentiation. Mechanical restitution and post-extrasystolic potentiation were plotted as

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the response of LV dP/dt to the two extrapulses. All LV dP/dt values were normalized to LV dP/dt during steady-state pacing. The influence of ryanodine on the heart rate dependent positive staircase ("Treppe") was investigated in six conscious dogs. Heart rate was controlled by left atrial pacing and increased in increments of 30 beats/min from 150 beats/min to 240 beats/min. The experiments were repeated with the end-diastolic diameter kept constant by volume loading with physiological saline solution (c. 2 0 0 - 1 0 0 0 ml 0.9% NaCI). Pacing at 240 beats/min could not be achieved in one of the six dogs.

Data analysis The data were recorded on a multichannel tape recorder (model 101, Honeywell, Denver, CO, USA) and displayed on a direct-writing oscillograph (Mark 200, Gould-Brush, Cleveland, OH, USA). Continuous recordings of LV dP/dt were derived from the LV pressure signal by use of operational amplifiers connected as differentiators with a frequency response of 700 Hz. A triangular wave signal was substituted for the pressure signals to directly calibrate the differentiator. LV end-diastolic dimensions were measured at the onset of LV contraction, indicated by the initial increase in LV dP/dt. LV end systole was defined as the point of maximum negative dP/dt. Ejection time (ET) was taken as the interval between maximum and minimum LV dP/ dt. LV isovolumic relaxation time was calculated using a three-constant exponential model of the LV pressure curve, fitted using a least-squares Levenberg Marquardt routine (Press et al., 1986). The isovolumetric time was taken as the interval starting with maximum negative dP/dt and continuing to a pressure which was 5 mmHg higher than the following end-diastolic LV pressure (Ihara et al., 1994). The beat-by-beat tau thus obtained was averaged over a total of 15 to 20 consecutive beats, to include a complete respiratory cycle. The percentage shortening of LV internal diameter was calculated with 100" (EDID-ESID)/EDID, where EDID is the enddiastolic and ESID is the end-systolic internal diameter. Wall thickening was calculated as ESWTEDWT, where ESWT is the end-systolic and EDWT is the end-diastolic wall thickness. Mean velocity of circumferential fiber-shortening corrected for heart rate (Vcfc) was formulated as (% shortening/lO0) *(x/60/HR)/(ET) (s - m ) where FIR is in beats/rain and ejection time (ET) is in seconds.

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Statistics The hemodynamic data were compared with a repeated measures ANOVA with analysis of contrasts for individual dose comparisons. The mechanical restitution, post-extrasystolic potentiation and "Treppe" data were analysed using repeated measures ANOVA with two within factors, normal v ryanodine, and interval. Analysis of contrasts was performed for comparison of control v ryanodine at the individual intervals. Data are given as mean _+S.V..M. and considered significantly different with P
Results Effects of ryanodine on LV and systemic hemodynamics The effects of systemic infusion of ryanodine with cumulative doses ranging from 0.5-4 pg/kg were investigated in conscious, chronically instrumented dogs (n = 12). Figure 1 shows representative traces recorded at baseline and 10 min after infusion of 4 pg/kg ryanodine. The changes induced by ryanodine remained stable over a period of at least 2 h after infusion, while complete recovery to baseline values occurred within 24 h. All data presented were collected at 10 min after administration of the ryanodine dose. Systemic infusion of cumulative doses of ryanodine (0.5-4 pg/kg i.v.) in conscious dogs resulted in a dose-dependent depression of cardiac contractility. Ryanodine induced relatively minor effects on mean arterial pressure, which rose by only 6_+ 3 mmHg and heart rate, which increased by 21 _+5 beats/min, while LV end-diastolic diameter did not change. The increases in mean arterial pressure and heart rate were not observed under ganglionic and muscarinic blockade. LV dP/dt (Fig. 2, left panel; Table 1) decreased from a baseline of 3182 _+ 117 mmHg/s to a value of 1 8 2 7 - + 9 8 m m H g / s after ryanodine 4/2g/kg ( - 4 1 . 5 _+4.0%). Even when baseline LV dP/dt was already depressed by ganglionic and muscarinic blockades, infusion of 4 pg/kg ryanodine still reduced LV dP/dt by the same degree ( - 4 2 . 2 _+2.6%) (Fig. 2, right panel). Vcfc was calculated as an additional parameter to assess the negative inotropic effect of ryanodine in vivo. Vcfc fell from 1.13_+0.08 to 0.71_+0.08 s -u2 after ryanodine 4 #g/kg (Fig. 3; Table 1). Ryanodine also prolonged the LV isovolumic relaxation time constant, tan. In the eight conscious dogs, in which this was examined, with heart rate constant at 150 beats/

rain, ryanodine, 4pg/kg, increased 23.1_+1.4 to 34.1_+3.6, P
tau

from

Effect of ryanodine on LV mechanical restitution and post-extrasystolic potentiation To assess the action of ryanodine on cardiac excitation-contraction in vivo, mechanical restitution curves (Fig. 4) and post-extrasystolic potentiation curves (Fig. 5) of LV contraction were obtained before and after systemic infusion of ryanodine, 4 #g/kg (1l = 7). During the rising phase of the mechanical restitution curve a significant difference between control and ryanodine (P<0.05) was observed only at the shortest extrasystolic pulse interval of 250 ms (Fig. 5). With longer extrasystolic pulse intervals ( 4 5 0 - 9 0 0 ms) the control mechanical restitution curve displayed overshoot and reached a stable plateau around 115% of steady-state LV dP/dt, while the mechanical restitution curve after ryanodine 4 #g/kg showed a continuous decline. Control post-extrasystolic potentiation declined towards a stable plateau slightly above 100% of steady-state LV dP/dt at longer extrasystolic pulse intervals. After ryanodine 4 pg/kg, the decline of the postextrasystolic potentiation curve was accelerated and demonstrated a continuing decline at longer pulse intervals (Fig. 5).

Effects of ryanodine on the "Treppe" In six dogs heart rate was controlled by left atrial pacing and increased in increments of 30 beats/ min from 150 beats/min to 240 beats/min. In intact, conscious animals the heart rate increase from 150 to 240 beats/rain produced only a minor increase in LV dP/dt by 7.5_+2.1% from 3298___ 148 mmHg/s (Fig. 6 left panel; Table 2). After ryanodine 4 #g/kg the increase of LV dP/dt was significantly greater (P<0.05, i.e. by 43.1 __4.7% from a baseline of 2222 _+ 110 mmHg/ s. The absolute increments in LV dP/dt increase (A LV dP/dt) were also significantly greater (P<0.05) for all tested heart rates after ryanodine, i.e., when heart rate rose from 150 beats/min to 240 beats/min the absolute increase in LV dP/dt was 244__ 72 mmHg/s in the absence and 952_+ 111 mmHg/s in the presence of ryanodine. LV enddiastolic diameter decreased during the pacing protocol (Table 2) from 33.7_+ 1 . 7 m m at 150 beats/ min to 30.1 _+1.4 mm at 240 beats/min for control, and from 34.8_+1.7mm at 150 beats/min to

Effects of Ryanodine on Cardiac Contraction

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Figure 1 Representative recordings of LV function and hemodynamics before (baseline) and 10 min after i.v. infusion of ryanodine, 4/~g/kg. Phasic measurements of left ventricular (LV) pressure, LV dP/dt, arterial pressure, LV internal diameter and wall thickness are shown.

30.8 _+ 1.6 mm after ryanodine 4 #g/kg. In another series of experiments the decrease in LV end-diastolic diameter during the pacing protocol was counteracted by volume loading with physiological saline solution (c. 2 0 0 - 1 0 0 0 ml 0.9% NaC1). With LV end-diastolic diameter constant (Table 2) the heart rate-dependent increase of LV dP/dt was still more pronounced after infusion of ryanodine 4. #g/kg (Fig. 6, right panel), when LV dP/dt rose by 1338 + 96 mmHg/s (65.5 + 5.3%) with increasing heart rate, and significantly less (P<0.05) by 896_+ 154 mmHg/s (27.3 +__4.9%) in the absence of ryanodine.

Discussion The present study demonstrated a dose-dependent negative inotropic effect of ryanodine in conscious, chronically instrumented dogs. Ryanodine also induced a negative lusitropic effect as reflected by

the increases in the isovolumic relaxation time constant, tau. The ryanodine dose range, in which this depression of cardiac contractility was observed (0.5-4/~g/kg), was significantly lower than the dosage used in the early studies of ryanodine effects in anesthetized dogs (_> 10/2g/kg), (Kahn et al., 1964:) and in the more recent study of Lappi and Billman (Lappi and Billman, 1993) in conscious dogs (10 #g/kg), but comparable to the lower doses used by Lew (Lew, 1993) in anesthetized dogs (1-16 #g/kg). In the lower dose ranges employed in the present study, ryanodine exerted little influence on preload and afferload in comparison with its pronounced depression of cardiac contractility. Ryanodine increased heart rate and mean arterial pressure modestly. The increase in mean arterial pressure was not observed after ganglionic blockade, thus indicating that it is more likely caused by an autonomic component rather than by a direct action on vascular smooth muscle. T h i s in, terpretation supports the findings of Procita eta/.

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Figure 2 Left panel: Dose-dependent effect of ryanodine (0.5-4 pg/kg, i.v.) on LV dP/dt in the conscious intact state (O, n= 12) and in the conscious state under ganglionic + muscarinic blockade ( 0 , n = 5). Right panel: LV dP/dt after infusion of increasing ryanodine doses, expressed as % depression from baseline before ryanodine.

Table I dogs

LV and systemic hemodynamics at baseline and after infusion of ryanodine 4pg/kg (i.v.) in 12 conscious

LV systolic pressure (mmHg) LV end-diastolic pressure (mmHg) Mean arterial pressure (mmHg) Heart rate (beats/rain) LV dP/dt (mmHg) Vcf~ (s -m) LV end-diastolic diameter (ram) LV end-systolic diameter (ram) % Shortening (%) LV end-diastolic wall thickness (mm) Systolic wall thickening (ram)

Baseline

Ryanodine (4 #g/kg)

A

1184-3 8.44-1 964-2 1054-5 3182 4-117 1.13 4-0.08 39.54-1.4 30.4+ 1.3 23.1 4-1.9 13.14-0.6 3.74-0.4

1144-3 6.94-1 1024-3 126-t-6 1827 4-98 0.71 _.+0.08 40.14-1.2 33.6_ 1.3 16.4 4-1.9 13.1-t-0.6 2.14-0.3

--4___3 --1.54-1 6__.3* 214-5" -- 1355 _ 163" --0.42 4-0.05* 0.64-0.6 3.2 4-0.4* -- 6.8 + 0.8* 0.04-0.1 --1.5-1-0.2"

Values are mean+_s.E.M. *P<0.05 (Procita et al., 1968) of a ryanodine-induced increase in peripheral resistance in anesthetized cats mediated by the central nervous system. The observed ryanodine induced heart rate increase was also reported by Lappi and Billman (1993). The mechanism of this heart rate increase is more difficult to understand. However, the increase in heart rate was not observed after ganglionic blockade, suggesting an autonomically mediated mechanism to the ryanodine-induced tachycardia. Clearly, the negative inotropic effect of ryanodine

is not autonomicaIIy regulated, as it still was observed to the same degree under ganglionic blockade. Mechanical restitution and post-extrasystolic potentiation of cardiac contraction are considered to reflect basic properties of excitation-contraction coupling in the heart (Banijamali et al., 1991; Cooper and Fry, 1990; RT.7.aheret al., 1992; Burkhoff et al., 1984; Yue et aI., 1985). The actions of ryanodine on mechanical restitution and postextrasystolic potentiation have been examined, in

Effects of Ryanodine on Cardiac Contraction 45

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Figure 3 Effectsof ryanodine (0.5-4 pg/kg, i.v.) on LV internal diameter (upper left), % shortening and Vcf~ (lower left), wall thickness (upper right), wall thickening (lower right), (n = 12, *P<0.05) (O % shortening; • Vcf~). vitro (Banijamali et al., 1991: Cooper and Fry, 1990; Ezzaher et al., 1992). The characteristic effects of ryanodine on mechanical restitution and postextrasystol!c potentiation have been attributed to its direct action on the SR Ca 2+ release channel, inducing continuous leakage of Ca 2+ from the SR (Banijamali et al., 1991; Cooper and Fry, 1990; Ezzaher et al., 1992). In the present study, ryanodine depressed both mechanical restitution and postextrasystolic potentiation curves, which is consistent with prior in vitro studies (Banijamali et al., 1991; Cooper and Fry, 1990; Ezzaher et al., 1992). In both cases greater effects of ryanodine were observed at longer extrasystolic intervals. As the interval between the last steady-state beat and the extrasystolic beat increases beyond the steady-state interval, the ryanodine-induced leakage of Ca -'÷ from the SR overcomes the re-uptake of Ca -'÷ into the SR, leading to a decrease in Ca -'÷ available for release and, thereby, to a decrease in contraction of the next, extrasystolic beat. At the same time, secondary intracellular mechanisms must buffer or sequester the Ca -'÷ extruded from the SR. This would explain the continuous decline of the mechanical restitution curve at longer extrasystolic pulse intervals. The decrease in releasable Ca -'+ for the

extrasystolic beat would also lead to a decreased Ca 2+ re-uptake, with the consequence of a weaker contraction of the following, post-extrasystolic beat, thus resulting in the observed continuous decline of the post-extrasystolic potentiation at longer intervals after ryanodine. Although the effects of ryanodine on mechanical restitution and post-extrasystolic potentiation are qualitatively similar in vivo and in vitro, some significant quantitative differences between the conscious dog and isolated cardiac muscle preparations were observed. Compared with the present data, the in vitro preparations are characterized by a much slower time course of the ryanodine effects (Banijamali et al., 1991; Cooper and Fry, 1990). These differences may be due to species or experimental conditions e.g., temperature and stimulation frequency in the in vitro experiments, as well as the inherent different contractile characteristics of in vitro preparations (Reichel, 1976). Another difference is that most in vitro studies demonstrated abolition of post-extrasystolic potentiation by ryanodine (F.~aher et al., 1992; Bose eta/., 1988; Wier and Yue, 1986; Schouten, 1990). Our results were less marked, particularly at the lowest intervals. These differences are due in part to 'the

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Figure 4 Mechanical restitution of LVdP/dt (normalized to levels during steady-state pacing) before (C)) and after (0) ryanodine 4pg/kg, i.v. ( n = 7 dogs, *P
Figure 5 Post-extrasystolic potentiation of LV dP/dt (normalized to levels during steady-state pacing) before ((3) and after (Q) ryanodine 4pg/kg, i.v. (n=7 dogs, *P
differences in vivo v in vitro in achievable pacing intervals. Another point of controversy between conscious animal experiments and isolated preparations is the physiological significance of the "Treppe." Since the classical study of Bowditch (Bowditch, 1871), numerous studies have demonstrated marked positive inotropic effects of increasing the frequency of contraction in excised myocardial strips (Blinks and Koch-Weser, 1961; Dale, 1932; Sonnenblick, 1962), isolated hearts (Monroe and French, 1961), and in situ hearts of anesthetized preparations (Boerth et al., 1969; Covell et al., 1967; Furnival et al., 1970; Lendrmn et al., 1960; Mitchell et a/., 1963). In contrast, most studies in conscious animals have reported a comparably insignificant inotropic effect of increased frequency on cardiac contraction (Higgins et al., 1973; Noble et al., 1969), which was also observed for intact, conscious dogs in t h e present study. Studies in conscious animals demonstrating a more significant influence of the "Treppe" are generally characterized by depressed baseline levels of LV dP/dt (Freeman et al., 1987). In the healthy, conscious dog baseline LV dP/dt is

approximately 3000 mmHg/s, as was observed in the present study. If baseline levels of LV dP/dt are depressed, it would be expected that a greater inotropic effect of the "Treppe" would be seen (Higgins et al., 1973). The controversy is actually more complex, due to different indices of contractility used, and different levels of heart rate studied (Maughan et al., 1985; Suga et al., 1983; Minra et a/., 1992). However, other than the study by Higgins et al., (1973) the same measurements were not compared in the same animals awake and anesthetized over the same heart rate ranges. Therefore, in the current investigation, as was done in the study by Higgins et al., (1973) we compared the same animals with and without ryanodine over the same heart rate ranges using the same indices of contractility. It has been suggested that the lack of major frequency potentiation in the conscious animal is, at least partially, caused by the counteracting effect of a decrease in preload, i.e., LV end-diastolic volume, which normally accompanies an increase in heart rate (Arentzen et al., 1978). In the present study the effects of ryanodine on the "Treppe" were

Effects of Ryanodine on Cardiac Contraction LV diameter decreasing

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Figure 6 Left panel: heart rate dependent increase of LV dP/dt (expressed as % of the LV dP/dt value at heart rate 150 beats/min) in the absence (O) and in the presence of ryanodine 4 pg/kg, i.v. (0). Right panel: LV dP/dt increase in the same dogs with LV diameter kept constant by volume load (n = 6 dogs, except for the 240 beats/min values, where n= 5 dogs. *P
Table 2

"Treppe" before and after infusion of ryanodine 4 pg/kg Changes from Baseline at:

LV diameter decreasing LV dP/dt (mmHg/s) control Ryanodine LV end-diastolic diameter (mm) control Ryanodine LV diameter*constant LV dP/dt (mmHg/s) control Ryanodine LV end-diastolic diameter (mm) control Ryanodine

Baseline 150 beats/min

210 beats/rain

240 beats/min

(n = 6) 3298 _+148 2222 _+110

01= 6) 164___108 610 _+84*

(n = 5) 244+ 72 952 _+111"

33.7_+1.7 34.8 _+1.7

-2.5___0.4 - 2.7 _+O.1

(n = 6) 5267_+213 2006 _ 98

(n = 6) 336_+77 779 -+ 57*

(n = 5) 896_+154 1338 -+96*

33.2-+ 1.6 34.4_+ 1.5

0.7-+0.3 0.0_+ 0.1

0.7_+0.2 --0.3_+ 0.2

-3.6_+0.4 - 4.0_+ 0.2

Values are mean_+s.~.M. *P
high basal inotropic state compared with more isolated preparations, we conclude that the normally insignificant "Treppe" in the conscious animal becomes more pronounced under conditions of depressed baseline contractility caused by impaired SR Ca-"+ handling. The existence of depressed contractility and alterations in SR Ca2+ handling have also been reported for isolated muscle preparations (Schouten et al., 1990). Therefore, it can be speculated that the classical Bowditch "Treppe" may reflect a state of myocardial depression due to alteration in SR Ca2+ handling, which is manifest in open-chest anesthetized and isolated muscle preparations, but that the "Treppe" plays a lesser role

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B. Kalthof et al.

in the physiological control of contraction in the normal heart. One explanation is that the enhanced 7+ "Treppe" is due to a different origin of Ca- , i.e., trans-sarcolemmal Ca-'+ v SR Ca 2+. In view of the ~+ effects of ryanodine on SR Ca- , it is possible that the trans-sarcolemmal Ca 2+ influx is enhanced with the "Treppe" in the presence of ryanodine. These differences as well as the effects of ryanodine on inotropy and lusitropy, suggest that altered SR Ca -'+ handling is one potential mechanism to explain depressed baseline levels of contractility in anesthetized and isolated muscle preparations. The conclusions of the present study must be tempered by the fact that the full range of the "Treppe" was not examined, i.e., only increases in heart rate from 150 to 240 beats/min. This was due to the elevation in baseline heart rate induced by ryanodine. Nonetheless, over this range of increasing cardiac frequency, ryanodine induced a marked potentiation of the "Treppe," which is the major finding of the current investigation. In summary, this study shows that ryanodine is a useful tool to investigate certain aspects of excitation--contraction coupling in conscious dogs. The results of this investigation indicate that signitlcant quantitative differences may exist between excitation-contraction coupling in the conscious animal and the situation previously reported for anesthetized, open-chest and isolated muscle preparations. These differences were most apparent with the "Treppe," which may help resolve the controversy that has existed regarding its influence in conscious animals compared with experiments in more isolated preparations.

Acknowledgement Supported in part by US Public Health Service grants HL 33107, HL 33065, HL 3 8 0 7 0 and RR 00168.

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