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University Microfilms International 300 North Zeeb Road Ann Arbor, Michigan 48106 USA St. John's Road, Tyler's Green High Wycombe, Bucks, England HP10 8HR BOUCHER, D.V.M., John Holly, 1930- INOTROPIC AND CHRONOTROPIC EFFECTS OF SOTALOL IN CONSCIOUS DOGS: ASSESSED IN COMBINATION WITH ATROPINE. The Ohio State University, Ph.D., 1977 Veterinary Science

Xerox University Microfilms,Ann Arbor, Michigan 48106 INOTROPIC AND CHRONOTROPIC EFFECTS OF SOTALOL IN CONSCIOUS DOGS: ASSESSED IN COMBINATION WITH ATROPINE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By John Holly Boucher, D.V.M., M.S.

* * * * *

The Ohio State University 1977

Reading Committee: Approved by Dr. Robert L. Hamlin Dr. Jean Powers Dr. Thomas E. Powers

Department of Veterinary Physiology and Pharmacology ACKNOWLEDGMENTS

I am grateful for the continuous encouragement and support given me by Professor Robert L. Hamlin. His con­ structive criticisms and patience were of immeasurable help in the completion of this work. To Adelbert L. Stagg, the most skillful, diligent, and devoted technical assistant I have ever known, I give my sincere thanks. I hold in my memory the lessons he taught me about the goodness of service to others. A special word of thanks to Ms. Margie Maxwell for her secretarial assistance in organizing and coordinating my efforts with others at the University. I am indeed appreciative of the support given me by the Department of Veterinary Physiology and Pharmacology. Particularly, the statistical and analytical assistance given me by Dr. Jean Powers was appreciated. Finally, I am grateful to my daughters, Jennifer and Carrie who, at times, not so graciously but, nevertheless, who understandingly and patiently allowed a great deal of neglect in order for me to complete this work. I owe them a debt of love I can never return. VITA

December 15, 1930 .... Born: Kansas City, Missouri 195 6...... B.S. and D.V.M. , Missouri University, Columbia, Missouri

195 7...... M.S., Cornell University, Ithaca, New York

1968...... M.S., The Ohio State University, Columbus, Ohio

PUBLICATIONS Boucher, J. H., W. H. Zech, and A. L. Stagg. Effective beta-adrenergic receptor blockade with sotalol~~in the absence of myocardial depression. Federation Proceedings 30:228. WTT. Boucher, J. H., D. E. Hilmas, C. T. Liu, W. P. Czajkowski, and R. 0. Spertzel. Myocardial depression during Diplococcus pneumoniae infection in monkeys. Proc. Soc. Exp. Biol. Med. 145:112-116. T 9 7 T : Zech, W. H., J. H. Boucher, D. E. Hilmas, and R. 0. Spertzel. Myocardial contractility in conscious rhesus monkeys: A method for long-term study. Am. J. Vet. Res. 35:83-86. T 5 7 T :

FIELDS OF INTEREST Ventricular function in work performance. Clinical veterinary cardiology. TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... ii

VITA...... iii LIST OF T A B L E S ...... vi LIST OF FIGURES...... vii

INTRODUCTION ...... 1 LITERATURE REVIEW ...... 3 MATERIALS AND METHODS ...... 12 Animals...... 12 Drugs...... 13 Instrumentation ...... 16 Indices of LV Function...... 17 Percent Chronotropic Blockade...... 21 Experimental Design ...... 21 Isoproterenol Duration of Response .... 21 Isoproterenol Dose-Response...... 2 4 Atropine Dose-Response ...... 24 Sotalol Dose-Response...... 24 Effectiveness of Beta-Adrenergic Blockade . . 25 Heart Rate-Contractility Relationship . . . 25 Sotalol-Atropine Sequences ...... 26 Statistical Analysis...... 27

RESULTS...... 28 Isoproterenol Duration of Response . 28 Isoproterenol and Atropine Dose-Response . . 28 Sotalol Dose-Response...... 36 Effectiveness of Beta-Adrenergic Blockade . . 42

iv Page Heart Rate-Contractility Relationship .... 47 Sotalol-Atropine Sequences...... 47

DISCUSSION ...... 51 Sotalol Dose-Response ...... 51 Effectiveness of Beta-Adrenergic Blockade. . . 54 Heart Rate-Contractility Relationship .... 56 Sotalol-Atropine Sequences...... 57

SUMMARY AND CONCLUSIONS...... ■ . . 61 BIBLIOGRAPHY...... 64

i v LIST OF TABLES

Table Page 1. ANOVA for isoproterenol duration of response...... 31 2. ANOVA for isoproterenol dose-response . . . 37 3. ANOVA for atropine dose-response ...... 38 4. ANOVA for sotalol dose-response ...... 41

5. ANOVA for incremental doses of sotalol (combined with atropine) relating graded doses of isoproterenol to percent chrono­ tropic blockade...... 45

6. Heart rate response to isoproterenol and sotalol + atropine (0.2 mg/kg)...... 46 7. Heart rate and corresponding myocardial contractility indices during graded atropine dosage and atrial pacing...... 48 8. Chronotropic and inotropic responses following a series of sequential dosings with sotalol and atropine...... 49 9. ANOVA for sotalol-atropine sequences. . . . 50

i vi LIST OF FIGURES

Figure Page 1. Manometer placement and cable modification . . 15 2. Estimation of maximal velocity of contractile element shortening (Vmax). 20 3. Definition of percent chronotropic blockade. . 23

4. Duration of inotropic and chronotropic responses to an intravenous bolus adminis­ tration of a 0.3 yg/kg dose of isoproterenol . 30 5. Graded dose-response curve for isoproterenol . 33 6. Graded dose-response curve for atropine . . . 35

7. Families of dose-response curves for sotalol (A: 0.5-4.0 mg/kg; B: 8.0 and 16.0 mg/kg) combined with atropine (0.2 mg/kg) relating the intrinsic heart rate (after autonomic blockade) to t i m e ...... 40

8. Family of dose-response curves for sotalol (0.5-16.0 mg/kg) combined with atropine (0.2 mg/kg) relating graded isoproterenol dosage to percent chronotropic blockade ...... 44

vii INTRODUCTION

The stimulatory effects of beta-adrenergic activity and the inhibitory effects of vagal activity on the ventric­ ular myocradium have been well documented (3). Autonomic reactivity and cardiovascular control mechanisms behave differently in conscious dogs than in those given anes­ thesia (63). The reasons for the differences are not yet understood, but, because differences do exist, traditional physiological mechanisms established under conditions other than consciousness should be re-evaluated in conscious subjects. Sinus arrhythmia is an arrhythmia resulting from waxing and waning of vagal efferent activity (67) , and is one autonomic phenomenon that operates in consciousness as well as during anesthesia.

Chemical denervation, to exclude the autonomic endog­ enous neuro-transmitter effects, presents the investigator with an excellent model to identify intrinsic myocardial alterations imposed by an intervention, i.e., responses would reflect a direct effect on the myocardium. Sotalol, a relatively new beta-adrenoceptor blocking drug, has been adequately characterized in anesthetized dogs (8, 19, 48).

The purpose of this investigation was to characterize the

1 2 inotropic and chronotropic effects of sotalol in conscious dogs when this beta-adrenoceptor blocking drug was com­ bined with atropine. LITERATURE REVIEW

The concepts of properties of myocardium (rhythmicity, excitability, conductivity, and contractility) (3) need no introduction for they have been recognized and discussed for many years. The first three properties deal with the electro-chemical functions of the myocardial cell, while contractility, a mechano-chemical process, involves excitation-contraction coupling of the myofibrils, i.e., the process by which actin and myosin interdigitate and shorten the fiber or generate tension. Methods for measur­ ing contractility in the intact heart have been the subject of intensive investigation. Sarnoff's studies initiated the contemporary era of studies in ventricular function with his description of homeometric autoregulation (54) , and Sonnen- blick extended his efforts to elucidate myocardial muscle mechanics (57). All four myocardial properties are affected potently by autonomic influences. For instance, increased sympa­ thetic activity augments norepinephrine release which stimu­ lates the myocardium mediated through beta-adrenoceptors located in both the atria and ventricles. The properties are thus affected in a positive sense. Other sympathetic 4

agonists include epinephrine from the adrenal medulla, and, a synthetic agent, isoproterenol. In contrast, in­

creased parasympathetic activity augments acetylcholine

release producing an inhibitory myocardial effect medi­ ated through the cholinergic receptors located primarily in the atria, with lesser numbers in the ventricles; the properties are thus affected in a negative sense. Com­ monly used antagonists, propranolol (a beta-adrenergic blocker) and atropine (a cholinergic blocker) are com­ petitive receptor blocking agents for their respective receptors. Contractility, the most elusive fundamental property of the myocardium to scientific investigation, is measured

as the estimated maximal velocity of fiber shortening occurring at zero load (57). It is independent of preload, afterload, and heart rate (59), excluding both Anrep (54) and Bowditch (7) effects. However, when afterload is in­ creased, contractility may increase (54) because of auto- regulatory mechanisms (Anrep effect) yet unknown, but postu­ lated to require increments in intracellular calcium ion concentration (33). Perhaps the most time-honored index of

contractility in the intact subject was been the first derivative of intraventricular pressure, dP/dt (39, 49). Various investigators also have used modifications of dP/dt

for contractility indices (39, 46, 55). dP/dt was used to 5

generate the calculated parameter, velocity of contractile elements (Vce) (40). Vce was used to construct a pressure- velocity curve for extrapolation to zero load, thus obtain­ ing the theoretical maximal velocity of contractile ele­ ment shortening (Vmax) (40). Sonnenblick, on the basis of isolated papillary muscle studies, presented evidence that contractility changes correlate most closely with the changes in Vmax (10, 57). He has maintained that Vmax is not influenced by changes in preload and afterload (59). This concept, compatible with the sliding filament concept of muscle contraction, has become widely accepted, and Vmax is considered by many to constitute the standard by which all other measurements of contractility should be judged (46). Others, however, have questioned its validity on theoretical grounds (41, 47). Of the potential determinants of contractility (pre­ load, afterload, inotropic state, and heart rate), the Bowditch effect (heart rate) remains an enigma. Bowditch

(7), in 1871, recognized that increased stimulation fre­ quency to the frog heart augmented its intensity of con­ traction, i.e., an increase in heart rate exerts an increase in myocardial contractility. This positive relationship between heart rate and inotropy has been duplicated, using standard contractility measurements, in the intact heart of anesthetized man (11, 58) and dog (12, 51, 65), and in 6 isolated heart muscle preparations (20, 32, 57). Con­ versely, in the only studies using conscious animals, Noble (42, 43) could not demonstrate the Bowditch effect; Higgins et al. (21) reported only a slight Bowditch effect in conscious dogs which became significantly greater when the same dogs were anesthetized. Both investigators felt the conscious subject responds differently from either anesthetized subjects or heart muscle preparations. Although not all the physiologic processes involved in the Bowditch phenomenon are clear, it is agreed that the calcium ion has a crucial role in the process (33). Upon cardiac cell membrane excitation, calcium is mobilized intracellularly; it binds with and blocks tropomysin which interferes with myosin-actin interaction, thus allowing the sliding filament interaction to operate at a velocity pro­ portional to the calcium concentration. When the interval between beats decreases (tachycardia), intracellular millieu restitution may be incomplete, and the accumulating calcium increases contractility by increasing troponin inhibition. Brooks et al. (8) showed the older hypothesis linking increased endogenous beta-adrenergic drive to increments in heart rate was inoperative. In anesthetized dogs with paced hearts, the Bowditch effect persisted even after beta- adrenergic blockade. 7

Since Ahlquist’s classic paper (1), in 1948, defin­ ing the dual theory of sympathetic receptors, alpha and beta, an explosion of new information dealing with adren­ ergic physiology and its associated pharmacology occurred.

No longer does the cardiovascular physiologist need to agonize over unexpected responses, contradictory to the supposed action of an intervention. He now has the knowl­ edge and the pharmacological tools to isolate and identify specialized autonomic mechanisms. As these new drugs be­ come available to the clinician, he too can shunt his frustrations of symptom treatment by better management of the mechanisms associated with adrenergically-based diseases. Our rapidly expanding knowledge attendant to the therapeutic importance of beta-adrenoceptor blocking drugs has led to their increased clinical usage to pre­ vent arrhythmias (17, 25, 50), to spare myocardial oxygen utilization (45), to reduce hypertrophy (15) , and to treat hypertension (23). The drug, propranolol, universally used for reducing these catecholamine mediated maladies, has a significant cardio-depressant action presumably due to its accompanying quinidine-like effect. In clinical situations where impingement upon myocardial contractility would compound an existing medical problem, propranolol must be used cautiously. Consequently, there has been an 8 intense search in the past decade for a beta-adrenergic blocker with less primary myocardial depressant activity. One such new agent, sotalol, DL-4-(2-isopropylamino-l- hydroxyethyl) methane sulphonanilide HC1, has been shown by Gomoll and McKinney (19) to be less cardiodepressive than propranolol in equipotent doses. Studies from several laboratories demonstrate sotalol to be an effective beta- adrenergic blocking agent in both experimental animals (6, 19, 36, 37, 48) and humans (9, 61). The issue of sotalol's effect on inotropy has been controversial. In isolated heart muscle preparations, sotalol has been found to be free from myocardial depres­ sant effects by most investigators (36, 37, 60); however, Blinks (5) reported that sotalol does have a primary myo­ cardial depressant effect in vitro. Puri and Bing (48), using anesthetized dogs, observed that sotalol reduced not only heart rate, but also the force and velocity of con­ traction. From hemodynamic data, they concluded that this effect of sotalol could be explained on the basis of its beta-adrenoceptor blocking activity without involving a direct myocardial depressant action. Admittedly, however, they were aware that the design of their study did not allow for a definitive separation of the relative roles of direct myocardial depressant and the beta-adrenoceptor blocking effects. Using reserpinized, anesthetized dogs, 9

Hoffman and Grupp (22) showed that sotalol had a modest depressant effect on myocardial contractility; in re- serpinized animals, the decrease in contractility should represent only the direct action of the drug on the myo­ cardium, thus excluding any beta-adrenoceptor blocking effect. Gomoll and McKinney (19) presented data, obtained from anesthetized normal and catecholamine-depleted (reserpinized) dogs, indicating that for a given degree of negative chronotropic influence, sotalol was accompanied by a proportionately smaller negative inotropic component, i.e., the inotropic state was heart rate-dependent. Con­ sistent with Gomoll and McKinney's data, Brooks et al. (8) reported only negligible depression (less than 6%) of LV dP/dt max in anesthetized dogs with fixed heart rates following doses of sotalol (6 mg/kg) which exceeded by 3-fold an adequate beta-receptor blocking level. They pro­ vided further evidence for the lack of depressant actions of sotalol by their failure to detect either a significant downward shift in force-velocity curves or a decrease in the estimated Vmax. Equivalent observations regarding Vmax have been made by Boucher et al. (6) in the conscious dog follow­ ing amounts of sotalol from 0.5-16 mg/kg given intra­ venously. Frankl and Soloff (16) and Brooks et al. (9) observed that the variables they used to measure inotropy

reduced as a result of rate-dependency, and concluded that 10 sotalol had no depressant effect on cardiac function in man. In another study in man, Lawrie et al. (34) reported that sotalol has both a negative chronotropic and a lesser negative inotropic effect. Sotalol*s predominant action is chronotropic; it has a slowing effect on heart rate. Gomoll and McKinney (19) showed that heart rate was dose-dependent and decreased 23-54% in anesthetized dogs following sotalol doses from 0.5-16.0 mg/kg. Other investigations in dogs (6, 8, 35, 48) and man (9, 16) confirm their report.

Gomoll and McKinney (19) demonstrated sotalol*s effectiveness, at various dose levels, to block progres­ sively incremented tachycardias induced by graded doses of isoproterenol. Each dose of sotalol produced typical pat­ terns of inhibition, i.e., parallel shift to the right of successive dose-response curves, versus the positive chronotropism induced by isoproterenol.

Reports by all investigators on the hemodynamic effects of sotalol on dogs have been remarkably consistent (8, 19, 48). Mean arterial pressure, cardiac output, left ventricular minute work, and systolic ejection period were depressed equivalently by sotalol. The alterations in cardiac output and left ventricular minute work appeared to be exclusively rate-related since both stroke volume and stroke work remained unchanged from control after sotalol. 11

LVEDP was not affected by sotalol; whereas, peripheral resistance increased.

The cardiovascular alterations of sotalol, from the data in the literature, suggests that its potential to spare oxygen may be its most positive characteristic. Sotalol's capacity to lower myocardial wall tension and stress are suggested by the depression of minute work, systolic tension-time index, and external work, all in association with the reduction in heart rate. These parameters, altered by sotalol, operate in the direction to reduce myocardial oxygen demands.

The impressive properties of this beta blocking agent (viz., effective blocker, minimal myocardial depressant, and effective oxygen-sparer) make sotalol a likely thera­ peutic replacement for propranolol in diseases exagger­ ated by catecholamine embarrassment. MATERIALS AND METHODS

Animals Nineteen male mongrel dogs in good health and weigh­ ing 20-27 kg were trained to lie quietly in right lateral recumbency on foam cushions. These dogs were selected

from a larger group because they adapted well to training and could achieve a basal tranquility in the laboratory environment. The criteria used for a basal state was the attainment of a sinus arrhythmia, a normal rhythm for

quietly resting dogs (67), and at least 20% reduction in HR within 5 minutes after being placed on the cushion. When adaptability to training was assured, the dogs were anesthetized with thiamylal sodium and methoxyflurane. The heart and great vessels were exposed by a sterile thoracotomy through the fifth intercostal space and sus­ pended in a pericardial cradle. A calibrated high fidelity micromanometer (Model 1017, Dynasciences Corporation, Pasa­ dena, CA) was inserted into the left ventricle through a stab incision in the apex. The incision was then closed with a simple interrupted suture on one side of the exiting

cable. The micromanometer was maintained in position within the ventricle by two plastic cuffs around the cable 13 as shown in Figure 1. Pacing electrodes were sutured to the right atrium in 7 dogs. The electrical leads were passed subcutaneously to the dorsal surface of the neck where they were exteriorized. During the 2-week recovery period, the relaxation training was continued. Each dog received 3-4 weeks of training before it was used in an experiment. Six to eight dogs were used in each experiment. The experiments were performed in a quiet, dimly lighted room with the dog placed in right lateral recum­ bency on a foam-cushioned table. The dog's trainer attended the dog throughout the experiment to assure its comfort and tranquility. Animal restraint posed no problem. The dogs were allowed to lie quietly for at least 10 minutes before an experiment was begun.

Drugs

Drugs were injected through an indwelling catheter placed in a cephalic vein. The drugs used in this study were 0.1, 0.3, 1.0, 3.0 or 10.0 yg/kg isoproterenol (ISP), a beta-adrenoceptor agonist; 0.5, 1.0, 2.0, 4.0, 8.0 or

16.0 mg/kg sotalol* (S), a beta-adrenoceptor antagonist; and 0.2 mg/kg atropine (A), a cholinergic receptor antagonist. Saline injections were randomly interposed as controls. ISP was administered as a bolus dose intravenously and

*As used here "sotalol" refers to the hydrochloride salt. 14

Figure 1. Manometer placement and cable modification. Two silastic cuffs (0.125 inch i.d. x 0.25 inch o.d. x 0.187 inch wide) were placed around the cable. One abutted against the cable-side of the manom­ eter and was secured by a tightly placed suture around the cuff. After implanting the manometer into the left ventricle (LV), the second cuff was secured around the cable tangent to the epi- cardium. The endocardial cuff protected the endocardium from damage by the manometer, and the epicardial cuff maintained the manometer in constant position within the LV. Ao = aorta. MYOCARDIAL WALL

MANOMETER

SILASTIC CUFFS 16 recordings were made at exactly 25 seconds post-injection. Administration of the other drugs will be discussed in the experimental design section.

Instrumentation Left ventricular pressure (LVP) was recorded at full scale from an implanted solid-state pressure microtrans­ ducer. The excellent frequency response (natural frequency in excess of 3 kHz) assured accurate reproduction of the pressure pulse (70). The pressure microtransducer, inter­ faced with a signal conditioner (Model BE-3, Dynasciences Corporation, Pasadena, CA), were calibrated from 0 to

300 mm of Hg pressure (1 volt = 300 mm of Hg). Voltage and pressure were linear within + 2%. Voltage correspond­ ing to gain and zero conditions of the signal conditioner was measured, using a fixed-resistance, standard bridge network for recalibration after implantation. Recali­ bration of the transducer-signal conditioner system was performed at least once weekly. The electronic first derivative of LVP was obtained by active differentiation (Model 620 analog computer, Biotronex Laboratory, Inc., Silver Spring, MD) to obtain dP/dt. The overall frequency response of the recorder system, including transducer, differentiating amplifier and recorder was greater than 150 Hz. The differentiating circuit was calibrated by imposing a signal of constant and known slope from an 17 integrating amplifier and measuring the resulting response of the differentiator. The right atrium was electrically stimulated with a Grass S-8 stimulator (0.5 msec duration, 3-5 volts). Heart rate (HR) was measured from a lead II electrocardiogram. The LVP, dP/dt and electrocardiogram signals were recorded on a pressurized-ink recording sys­ tem (Brush Mark 200, Gould, Inc., Cleveland, OH), operated at paper speeds from 10 to 200 mm/second. During record­ ings for calculation of Vmax, paper speed was 200 mm/second. Zero pressure was estimated at a point near mid-diastole because the sealed manometer could not be referenced to atmosphere after implantation.

Indices of LV Function

Two indices of left ventricular function were used in this study: Maximal rate of rise of left ventricular pres­ sure (dP/dt max), and estimated maximal velocity of con­ tractile element shortening (Vnax). The latter parameter is widely accepted as a measure of myocardial contractility that is relatively independent of both preload and after­ load (40, 59); while the former parameter is dependent upon contractility, preload and to a much lesser extent on after­ load (39, 65). However, since preload, as measured by left ventricular end-diastolic pressure, did not change signifi­ cantly in these studies, any alteration in dP/dt max reflects either an alteration in contractility or afterload. 18

The Maxwell three-component version of the Hill model for muscle (71) was employed, using left ventricular iso- volumic developed pressure (DP) (62) (DP = LVP minus LVEDP), to estimate Vmax by the pressure-velocity method described by Mason (40). This model was adapted because it is insensitive to the artifacts introduced by base-line drift in the manometer (72) and to the inaccuracy of LVEDP estimation because, by the DP method, LVEDP represents zero pressure. Instantaneous velocity of contractile elements (Vce) was calculated at 5 msec intervals during isovolumic con­ traction as :

Vce = 32P^d'DP > where

32 is the coefficient of series elasticity (1/muscle lengths) at body temperature (73) and allows the calculation in terms of muscle lengths (ML) per second. A pressure- velocity curve relating Vce to the corresponding DP was constructed and the last 3-5 Vce calculations on the de­ scending limb were extrapolated, by least squares linear regression, to zero pressure to obtain the estimated Vmax (Figure 2). This Vmax represents the estimated velocity of contractile element shortening at zero load. 19

Figure 2. Estimation of the maximal velocity of contractile element shortening (Vmax). A: Representative recording of high fidelity left ventricular pres­ sure (LVP) and its first derivative (dP/dt) at paper speed of 200 mm/sec. Instantaneous points, at 5 msec intervals, between LVEDP and P0 were used to calculate the velocity of contractile elements (Vce). B: The descending limb of the developed isovolumic pressure (DP) vs. Vce relationship was extrapolated linearly to zero pressure to obtain the estimated Vmax. LVEDP = left ventricular end-diastolic pressure; P0 = maximal isovolumic pressure. dP/dt LVP (mm Hg/sec) (mm Hg) — ro o o o ^ o o o u» o 0 o o o o o o 1 » i ..-i L_ J I-- 1— u

IN) u o

VELOCITY OF CONTRACTILE ELEMENTS (MUSCLE LENGTHS/SEC) o < m n W ro o. ■D o TJ

3 PI5 n"O u CD 3 W _ z C «Q § 9° m r ® c o

o

03 21

Percent Chronotropic Blockade The parameter percent chronotropic blockade, as defined in Figure 3, describes the relative action of sotalol (given with 0.2 mg/kg atropine) in quantitative terms (74). The basal HR change due to ISP after auto­ nomic blockade was divided by the HR change due to the same amount of ISP in the absence of autonomic blockade. The ratio obtained derives the HR responsiveness to ISP in the presence of sotalol. By subtracting the HR re­ sponsiveness value from 1.0 and multiplying by 100, the calculated percent blockade to the ISP dose is obtained.

Experimental Design

Isoproterenol Duration of Response. To ascertain the duration of peak response and the total response du­ ration of ISP, 6 dogs were given a 0.3 yg/kg bolus dose intravenously. A control observation was followed by observations made for 2 minutes (5, 15, 20, 25, 30, 35, 45, 60, 90 and 120 seconds) after the ISP. Subsequent experiments utilized the duration of ISP response to deter­ mine the proper time to make a recording at peak response, and to determine an adequate interval between doses. Parameters measured were HR, dP/dt max and Vmax. The results were averaged and plotted against time. 22

Figure 3. Definition of percent chronotropic blockade. Example: the control heart rate change after isoproterenol (3.0 yg/kg) was 150 beats per minute. The same isoproterenol dose, after autonomic blockade (S + A), effected a heart rate change of 30 beats per minute, or, 0.20 of the control response. Percent blockade = 100 (1-0.20) = 80%. Dashed line = basal heart rate; AHR = change from basal heart rate; ISP = isoproterenol; S = sotalol; A = atropine. HEART RATE (B E A TS /M IN ) BLOCKADE = PERCENT 300-t 0 5 2 200 150 0 i 100 H i CONTROL T T ISOPROTERENOL ISOPROTERENOL 0 0 1 T - l ( a h r r h a . . 10 3.0 1.0 .3 .1 0 —//i / / f— ATROPINE (0.2 mg/kg) (0.2 ATROPINE mg/kg) SOTALOL(8.0 dueto (\iq/kq) + ---- 1 'sPwUhoutS+a) 1 ----- 1 ---- A HR(30) 1 ----

10.0 1 24

Isoproterenol Dose-Response. Following a control measurement, bolus injections of ISP (0.1, 0.3, 1.0, 3.0 and 10.0 yg/kg), spaced at 10 minute intervals, were given intravenously to 7 dogs. The chronotropic response was measured at maximal effect. The results were averaged and plotted semi-logarithmically.

Atropine Dose-Response. Following a control measure­ ment, three-minute infusions of A (0.02, 0.05, 0.1, 0.5 and 1.0 mg/kg) were given intravenously to 6 dogs. The chrono­ tropic response was measured 10 minutes after completion of the infusion. Successive doses were spaced at 2-day inter­ vals minimally. The results were averaged and plotted arithmetically.

Sotalol Dose-Response. Following a control measure­ ment, three-minute infusions of S (0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/kg) combined with A (0.2 mg/kg) were given intravenously to 7 dogs. The chronotropic response was measured each minute for 5 minutes starting at the comple­ tion of the infusion. Successive doses were spaced at 2-day intervals minimally. The same procedure was per­ formed on another group of 8 dogs at S doses of 8.0 and 16.0 mg/kg combined with A (0.2 mg/kg). The results were averaged and plotted against time. 25

Effectiveness of Beta-Adrenergic Blockade. S (0.5, 1.0, 2.0 and 4.0 mg/kg) combined with A (0.2 mg/kg) was tested for its beta-adrenergic blockade effectiveness against incremental doses of ISP in 6 dogs (Group A). Following a basal HR measurement in the control animal, successive bolus injections of ISP (0.1, 0.3, 1.0, 3.0 and 10.0 yg/kg), spaced at 10-minute intervals, were given intravenously. Ten minutes after the final ISP injection, a 3-minute infusion of S + A was given intravenously. Ten minutes after the S + A infusion had begun, the ISP chal­ lenge doses were repeated. Successive doses of S were spaced at 2-day intervals minimally. The same procedure was performed on another group of 8 dogs (Group B) for S (8.0 and 16.0 mg/kg) combined with A (0.2 mg/kg), but challenged with ISP doses of 0.3 and 1.0 mg/kg only. Again, the same procedure was performed on another group of 7 dogs (Group C) which received the same S + A doses as Group B dogs, but were challenged with 3.0 yg/kg of ISP only.

At each ISP dose level and for each S dose, percent chronotropic blockade was calculated, the results were averaged, and a family of dose-response curves were plotted.

Heart Rate-Contractility Relationship. To study the influence of HR on the indices of LV function (dP/dt max and Vmax), HR was increased from the basal state by two 26 methods devoid of adrenergic activity: (1) atropine- induced increments and (2) atrial pacing. In both studies, HR was calculated by the interval-between-beats (using the interval previous to the waveform used for contractility determinations). A (0.02, 0.05, 0.1, 0.5, and 1.0 mg/kg)

(amounts that increased HR from 10 to 96 beats per minute from the basal rate) was infused intravenously over a 3- minute period. Simultaneous chronotropic*and inotropic responses were measured 10 minutes after the drug infusion. Atrial pacing rates of 150, 170, 190 and 210 beats per minute (rates that increased HR from 38-98 beats per minute from the basal rate) were performed on 7 dogs. Simul­ taneous chronotropic and inotropic measurements were made at each HR increment. In both studies, a HR-contractility relationship was established for each dog, and a multiple regression analysis was employed to test the slope of the composite results.

Sotalol-Atropine Sequences. S (8.0 mg/kg) and A (0.2 mg/kg) was administered in alternating sequences and in combination to 6 dogs to determine if order of adminis­ tration has any consequences on chronotropic or inotropic (dP/dt max and Vmax) effect. Following a control obser­ vation, a 3-minute infusion of S was given intravenously.

Ten minutes after the S infusion had begun, a recording was made, then a 3-minute infusion of A was given 27 intravenously. Ten minutes after the A administration had begun, a recording of the combined drug effect was made.

The drug order was reversed so that A was administered first, and the procedure was repeated. In the final procedure, a control observation was followed by a 3- minute infusion of S combined with A, and 10 minutes after the infusion had begun, a recording was made of the com­ bined effect. Results of all observations were averaged, graphed and tested.

Statistical Analysis To evaluate the results, analysis of variance (ANOVA) with repeated measures was performed on each appropriate study. This was followed by the Student-Newman-Keuls Test when significant differences were found. In addition, when applicable a Dunnet's t Test was done to compare treatment groups to the control group. If a significant interaction was found, a one-way ANOVA was then performed. Least squares linear regression was used to test the slope in the HR-contractility relationship studies. The 95% confi­ dence level was employed for significance. RESULTS

Isoproterenol Duration of Response. The peak re­ sponse in HR, dP/dt max, and Vmax occurred uniformally 15 through 35 seconds following a rapid intravenous injection (bolus) of ISP, 0.3 pg/kg (Figure 4). The ANOVA (Table 1) showed a significant difference between the control and the respective peak values for each parameter: heart rate (P < 0.001), dt/dt max (P < 0.001), and Vmax (P < 0.002). The sensitivity of dP/dt max at peak response was 138 percent above control, whereas, Vmax was elevated 15 percent. Data collection, always made at exactly 25 seconds following ISP injections, therefore, was within the peak period. The ISP half-life response (-.693/decay slope) for HR, dP/dt max, and Vmax (47.1, 31.5, and 31.8 seconds respectively), as calculated from the curves in Figure 4, was short enough so that no response was detectable 5 minutes after the in­ jection.

Isoproterenol and Atropine Dose-Response. Figures 5 and 6 show the dose-response curves for graded doses of ISP and A respectively. The ANOVA disclosed significant differ­ ences in heart rate between doses of both ISP (P < 0.001)

28 29

Figure 4. Duration of inotropic and chronotropic responses to an intravenous bolus administration of a 0.3 yg/kg dose of isoproterenol (ISP) (arrow). Each point represents the mean of observations on 6 dogs. The peak response duration for heart rate, maximal rate of rise of left ventricular pressure (dP/dt max), and the estimated maximal velocity of contractile element shortening (Vmax) was from 15 through 35 seconds after ISP. The asterisk indicates the time after ISP that measurements were taken. The insert shows the half-life (ti/2) response to ISP, in seconds, for each parameter. PERCENT CHANGE FROM CONTROL 120 I40i 60 40 20 80 — 0 l P tlS 30 SECONDS Vmax Pd max dP/dt ER RT 47.1 RATE HEART 90 tVj(sec) 31.8 31.5

120 Table 1. ANOVAa for isoproterenol duration of response.

Source of Degrees of Level of Variance Freedom Mean Square F Significance b HR responses 10 6,057.5 20.9 < 0.001 Subjects within responses 50 290.3 (Error) dP/dt max responses 10 21.0 31.7 < 0.001 Subjects within responses (Error) 50 0.66

Vmax responses*3 10 0.13 3.42 < 0.002 Subjects within responses 50 0.04 (Error)

a Repeated measures on the respective response groups for heart rate (HR), maximal rate of rise of left ventricular pressure (dP/dt max), and the estimated maximal velocity of contractile element shortening (Vmax). 1. The respective parameter responses were measured at 0, 5, 15, 20, 25, 30, 35, 45, 60, 90, and 120 seconds. 32

Figure 5. Graded dose-response curve for isoproterenol. Each point represents the mean of the obser­ vation of 7 dogs where standard error (S.E.M.) is shown by the horizontal bars. Dosages are plotted on the horizontal axis. O j < LO 1 ------1 \ 0-1 • ------I ______I ______I

______

ro ro ■t±o> co ro ro o o o _1 h002 HEART RATE (beats/minute)

ISOPROTERENOLOug/Kg) 34

Figure 6. Graded dose-response curve for atropine. Each point represents the mean of the observation of 6 dogs whose standard error (S.E.M.) is shown by the horizontal bars. Dosages are plotted on the horizontal axis. 220-1

u 200 -

180-

“ 160-

4 0 -

20 -

100-

0 0.02 005 oT ATROPINE, mg/kg 36

(Table 2) and A (P < 0.001) (Table 3) confirming a graded dose-response. Using 10.0 yg/kg of ISP as the comparison dose, Dunnet's t Test showed no significant difference between the comparison dose and the 3.0 yg/kg dose, whereas, the remaining doses were significantly different from the 10.0 yg/kg dose (P < 0.005). Therefore, the mean HR for ISP reached an apparent asymptote by the 10.0 yg/kg dose.

Sotalol Dose-Response. Figure 7 shows a plot of heart rate vs. time for dogs receiving graded doses of S with a fixed dose of A. To all doses of S plus A, heart rates accelerated.

The heart rates after S doses of 0.5-4.0 mg/kg (Figure 7A) were examined by .ANOVA (Table 4). The signifi- cent interaction between dose and time was investigated further by doing a one-way ANOVA for each dose level to determine if there was a significant difference between times within that dose. This was followed by a Dunnet's t Test to determine at which time the HR deviated signifi­ cantly from the control values. At doses of 0.5, 1.0, and 2.0 mg/kg all time intervals differed significantly from their respective control. However, no differences were found among the times at a dose of 4.0 mg/kg. After a one-way ANOVA, Dunnet's t Test indicated that the treatment heart rates increased significantly from Table 2. ANOVAa for isoproterenol dose-response.

Source of Degrees of Level of Variance Freedom Mean Square F Significance

Doses^ 5 107,624.0 181.2 < 0.001 Subjects within doses 30 594.1 (Error) 0SDc 3 2,028.4 2.7 >0.05 Subjects within OSD 18 746.4

Doses x OSD 15 343.1 2.5 < 0.003 Residual 90 134.9

aRepeated measures on doses and OSD.

0.0, 0.1, 0.3, 1.0, 3.0, and 10.0 ug/lcg doses of isoproterenol.

Observations per subject per dose. Table 3. ANOVAa for atropine dose-response.

Source of Degrees of Level of Variance Freedom Mean Square F Significance

Doses*5 5 11,599.4 29.1 < 0.001 Subjects within doses 25 399.2

aRepeated measures on doses.

0.0, 0.02, 0.05, 0.1, 0.5, 1.0 mg/kg doses of atropine.

w 00 39

Figure 7. Families of dose-response curves for sotalol (A: 0.5-4.0 mg/kg; B: 8.0 and 16.0 mg/kg) combined with atropine (0.2 mg/kg) relating the intrinsic heart rate (after antonomic blockade) to time. Each point represents the mean of observations of 7 dogs (Group A) and 8 dogs (Group B). The stippled area represents a 3-minute intravenous infusion of the designated amounts of sotalol (S) plus atropine (A). C = control observation. 8 0 i S + A S + A • 0.5mg/Kg

170 .Omg/Kg 16.0 mg/Kg

E 160 2.0 mg/Kg 8.0 mg/Kg 150 • 4.0 mg/Kg

140 x 130 • GROUP A (7) o GROUP B (8)

120-

— I..... ffn -1---- r----- 1---T--- 1 -3 0 I 2 3 4 5 C MINUTES C MINUTES •P' o Table 4. ANOVAa for sotalol dose-response.

Source of Degrees of Level of Variance Freedom Mean Square F Significance

Doses^ 3 7,238.5 8.38 < 0.001

Subjects within doses 18 863.7 (Error)

Time 6 5,490.8 5.87 < 0.001 Subjects within time 36 935.9

Doses x Time 18 203.8 2.57 < 0.001 Residual 108 79.3

aRepeated measures on doses and time.

^0.5, 1.0, 2.0, and 4.0 mg/kg doses of sotalol. 42 control heart rates at S doses 8.0 (P < 0.005) and 16.0 rog/kg (P < 0.005) (Figure 7B). A trend indicating a "re­ bound" effect in dose-response appeared with doses greater than 4.0 mg/kg. The augury of a drug-receptor alteration is strengthened by the heart rate instability of the 16.0 mg/kg S dose; heart rates at all other S dosages were relatively stable.

Effectiveness of Beta-Adrenergic Blockade. A family of dose-effect curves was constructed by relating the per­ centage chronotropic blockade for a wide range of S doses plus a fixed dose of A to incremental doses of ISP (Figure 8). S displayed its antagonism to the exogenously elicited adrenergic response by near complete blockade to lower doses of ISP. The ANOVA (Table 5) demonstrated that a significant drug interaction (P < 0.004) occurred and accounted for the atypical patterns of inhibition, i.e., nonparallelism, and convergence of the curves at high ISP doses. The ANOVA also disclosed significant differences between doses for S (P < 0.008) and ISP (P < 0.001), indi­ cating that an increased dosage caused a graded response.

Table 6 displays the graded response and shows that heart rate increased with increments of ISP for a given amount of S; likewise, heart rate generally decreased with increasing amounts of S for a given amount of ISP. 43

Figure 8. Family of dose-response curves for sotalol (0.5-16.0 mg/kg) combined with atropine (0.2 mg/kg) relating graded isoproterenol (ISP) dosage to percent chronotropic blockade. Each point represents the mean of observations on 6 dogs (Group A), 8 dogs (Group B), and 7 dogs (Group C). ISP dosages are plotted on a hori­ zontal axis. Percent blockade for 8.0 and 16.0 mg/kg doses of sotalol were calculated at ISP doses of 0.3, 1.0, and 3.0 yg/kg only. PERCENT CHRONOTROPIC BLOCKADE 100 80- 40- 60-j 20 0J - - 4.0mg/kg 2.0mg/kg 0.5 mg/kg0.5 I .Omg/kg RU A (6) A • GROUP GOP (8) B oGROUP GOP (7) CAGROUP OAO TOIE 02 g/kg) SOTALOL (0.2m + ATROPINE

. 03 . 3.0 1.0 0.3 0.1 SPOEEO (jug/Kg) ISOPROTERENOL

‘ ^ 8.0 mg/kg 8.0 ^ a 16.0 mg/kg lo.o

44 Table 5. ANOVAa for incremental doses of sotalol (combined with atropine) relating graded doses of isoproterenol to percent chronotropic blockade.

Source of Degrees of Level of Variance Freedom Mean Square F Significance

Sotalol (S) doses^ 3 2,515.1 5.68 < 0.008 Subjects within S doses 15 443.2 (Error)

Isoproterenol0 (ISP) doses 4 13,531.9 26.1 < 0.001

Subjects within ISP doses 20 519.5

S doses x ISP doses 12 226.6 2.84 < 0.004 Residual 60 94.0

Repeated measures on S and ISP doses.

O.S, 1.0, 2.0, and 4.0 mg/kg doses of sotalol (combined with 0.2 mg/kg atropine).

c0.1, 0.3, 1.0, 3.0, and 10.0 yg/kg doses of isoproterenol. 46

Table 6. Heart rate response to isoproterenol and sotalol + atropine (0.2 mg/kg).a

Isoproterenol (yg/kg) Sotalol (mg/kg) Control 0.1 0. 3 1 .0 3.0 10.0

Control 124 (4)b,C 189 (4) 220 (4) 253 (3) 278 (4) 289 (5) 0.5 184 (14) 197 (15) 231 (12) 263 (10) 283 (6) 298 (5)

1.0 179 (10) 186 (15) 206 (12) 245 (11) 270 (7) 289 (4)

2.0 162 (13) 164 (13) 183 (11) 221 (12) 248 (10) 283 (7) 4.0 148 (12) 150 (12) 164 (14) 186 (15) 222 (14) 254 (12) 8.0 148 (10) 159 (14) 176 (13) 176 (18)

16.0 150 (10) 170 (14) 176 (13) 156 (10)

aThree groups of dogs were used in this study: Group A = 0.5-4.0 mg/kg doses of sotalol at all isoproterenol doses (n = 6). Group B* = 8.0 and 16.0 rag/kg doses of sotalol with 0.3 and 1.0 yg/kg doses of isoproterenol (n = 8). Group C* = 8.0 and 16.0 mg/kg doses of sotalol with 3.0 yg/kg of isoproterenol (n = 7). *The control values of the Groups B and C dogs were pooled. Therefore, calculations of percent block­ ade from the values in this table at 8.0 and 16.0 mg/kg doses of sotalol do not duplicate the points at these doses in Figure 8, nor do the intrinsic heart rates (after autonomic blockade) duplicate Figure 7.

bMean beats per minute (+ S.E.M.).

The control/control value was obtained from Group A dogs at 4 observations per dog. 47

Heart Rate-Contractility Relationship. When heart rates of conscious dogs were increased by atrial pacing (150-210 beats per minute) or by atropine doses (0.02, 0.05, 0.1, 0.5, and 1.0 mg/kg), no significant relation­ ship between HR and either dP/dt max or Vmax existed (P > 0.05). Table 7 summarizes the results of mean heart rates and their corresponding mean contractility indices.

Sotalol-Atropine Sequences. Table 8 shows the mean

(+ S.D.) values for HR, dP/dt max, and Vmax at control periods and after alternating sequential administrations of S (8.0 mg/kg) and A (0.2 mg/kg). The ANOVA revealed significant differences (P < 0.001) among the HR groups

(Table 9). Results of the Student-Newman-Keuls Test (SNK) showed that A, given alone, evoked a HR response that was significantly different from all the other groups (P < 0.001). There were no other significantly different HR groups. Although ANOVA revealed significant differences (P < 0.007) among the dP/dt max groups (Table 9), it was not of sufficient strength for SNK to discriminate changes.

Significant differences were not found among the Vmax groups. 48

Table 7. Heart rate and corresponding myocardial contrac­ tility indices during graded atropine dosage and atrial pacing.

Heart Rate dP/dt max Vmax (bpm) (mmHg/sec) (ML/sec)

Atropine (mg/kg): Control 6 116 (2) 3850 (204) 2.06 (0.02) 0.02 6 126 (1) 3500 (184) 2.11 (0.03) 0.05 6 186 (6) 3450 (166) 2.10 (0.04) 0.1 6 208 (4) 3450 (335) 2.06 (0.07) 0.5 6 209 (4) 3230 (274) 2.12 (0.07) 1.0 6 212 (4) 3320 (188) 2.15 (0.04)

Atrial Pacing: Control 7 112 (6) 3625 (367) 2.33 (0.14) PR 7 150^ 4170 (542) 2.38 (0.12) PR 7 170 4243 (601) 2.26 (0.12) PR 7 190 4401 (551) 2.29 (0.13) PR 7 210 4690 (606) 2.28 (0.19)

aMean (+ S.E.M.).

^This is a pacing rate (PR), therefore, there is no variance. N = Number of observations. bpm = Beats per minute.

dP/dt max = Maximal rate of rise of left ventricular pressure.

Vmax = Estimated maximal velocity of contractile element shortening.

ML = Muscle lengths. 49

Table 8. Chronotropic and inotropic responses following a series of sequential dosings with sotalol and atropine.

Heart Rate dP/dt max Vmax (bpm) (mmHg/sec) (ML/sec)

Control 117 (18)c 3,650 (637) 2.24 (0.22) Sotalol CS) b 101 (11) 2,970 (539) 2.28 (0.29) Atropine (A) 141 (24) 2,780 (416) 2.29 (0.22)

Control 109 (20) 3,510 (956) 2.35 (0.25) Atropine 206 (2 2)*** 3,220 (416) 2.36 (0.34) Sotalol 127 (22) 2,650 (588) 2.34 (0.20)

Control 121 (22) 3,790 (1,029) 2.41 (0.20) S + A 146 (32) 2,730 (343) 2. 39 (0.25)

aSotalol, 8.0 mg/kg.

^Atropine, 0.2 mg/kg.

cMean (+ S.D.). bpm = Beats per minute.

dP/dt max = Maximal rate of rise of left ventricular pressure.

Vmax = Estimated maximal velocity of contractile element shortening. ML = Muscle length. *** = p < 0.001. Table 9. ANOVAa for sotalol-atropine sequences.

Source of Degrees of Level of Variance Freedom Mean Square F Significance

Expt'l. groups on HR 7 6,522.8 16.2 < 0.001 Subjects within groups 35 401.9 (Error)

Expt'l. groups on dP/dt max 7 1.19 3.4 < 0.007 Subjects within groups 35 0.35 (Error)

Expt'l. groups on Vmax 7 ’ 0.021 0.4 > 0.05

Subjects within groups 35 0.049 (Error)

Repeated measures on the respective groups for heart rate (HR), maximal rate of rise of left ventricular pressure (dP/dt max), and the estimated maximal velocity of contractile elements (Vmax). DISCUSSION

Figures 4 and 5, the time-response and dose-response curves respectively for ISP, confirm the sensitivity, du­ ration of response, and validity of the model used in this study to investigate chronotropic effects of drugs. They also establish, for the conscious dog, responses to a set of challenging doses of ISP. Under the conditions of this experiment, dP/dt max appeared to be a more sensitive index of inotropic change than Vmax. However, afterload change in response to ISP may have contributed to some of the change in dP/dt max and not Vmax. 0.2 mg/kg of A, the dose combined with all S doses, was 2-fold the vagolytic dose since maximal heart rates were reached by a 0.1 mg/kg dose (Figure 6). This dose also was one-third the amount which promoted CNS stimu­ lation and nicotinic effects, i.e., hyperexcitability, skeletal muscle tremors, and muscle weakness. Donald (14) confirms the vagolytic effectiveness of 0.2 mg/kg A in conscious dogs.

Sotalol Dose-Response. The well-known response that heart rate accelerates to a vagolytic dose of A is illus­ trated in Figure 7A. However, when S, in graded doses, was

51 52 added to the fixed dose of A, the degree of cardioacceler- ation was attenuated 35-65 beats per minute from the rate of a vagolytic dose of atropine (given alone) by increas­ ing doses of S. Figure 7B, however, shows that when ex­ tremely large doses (8.0 or 16.0 mg/kg) of S were given with A, the tachycardia induced by A was not attenuated so markedly as with lower doses of S. Why did the lower doses of S reduce the degree of cardioacceleration produced by a vagolytic dose of A? One explanation is that S stimulates parasympathetic receptors. This is unreasonable because A did not prevent the chrono­ tropic response. Alternatively, S may depress the sino­ atrial node directly. This proposal is consistent with

Singh and Vaughan Williams' (56) report demonstrating that

S prolonged the duration of the myocardial intracellular action potential. This is unlikely as the only mechanism since, as shown in Table 8, S given alone decreased the rate of sino-atrial discharge 16 beats per minute, whereas the same amount of S depressed an atropine-induced vagolytic rate 79 beats per minute.

Why did a heart rate "rebound" effect occur at the 8.0 and 16.0 mg/kg amounts of S, thereby, permitting the same degree of cardioacceleration to A as occurred with 2.0 mg/kg of S, and why was heart rate instability observed with 16.0 mg/kg of S? Such stimulation suggested that S 53 may have intrinsic sympathomimetic activity (ISA) in high doses similar to that reported for pronetholol (2). Al­ though S has never been reported to possess ISA, this finding may reflect an autonomic reactivity difference in conscious subjects not seen in anesthetized subjects. Investigators utilizing autonomic blockade by either chemical or surgical denervation of the heart have con­ sistently reported an increased heart rate after blockade (4, 28, 38, 44) just as was found in this study. The mechanism for this curious effect to autonomic blockade is not completely understood, but in a vast literature review, Jensen (26) explains that, under aneural conditions, cardiac rate is regulated by pressure-sensitive receptors in the myocardium, primarily in pacemaker tissue. He further sug­ gests that because of the narrower limits of heart rate change produced by the intrinsic regulating mechanism, its activity is obscured by the autonomic control. Jose (29, 30) demonstrated that the intrinsic heart rate (after propranolol and atropine) was highly correlated with myocardial contractility, and he used this parameter to predict the myocardial contractile status of human patients (27, 31). Jose reasoned that both intrinsic automaticity and contractility may be dependent upon some third common factor (energy source). 54

Effectiveness of Beta-Adrenergic Blockade. The family of curves relating S dosage to percentage chrono­ tropic blockade to incremental ISP doses, is a mirror-image of conventional dose-response curves: 100 per cent chrono­ tropic response to ISP is equivalent to zero blockade, and zero response to ISP is equivalent to 100 per cent blockade. Percent blockade, therefore, is the reciprocal of percent ISP responsiveness. On the basis that an adequate beta- adrenergic blockade represents at least an 80% inhibition

of an ISP dose (0.3 ug/kg) which produces a 95 beat per minute heart rate increase from control, then 2.0 mg/kg of

S given with A is an effective beta-adrenoceptor blocking dose. S doses of 8.0 and 16.0 mg/kg caused a near-complete beta-adrenergic blockade (93 and 96% respectively) to 0.3 ug/kg of ISP. Data consistent with these observations have been reported (19) also for S given alone in anes­ thetized dogs, thus confirming that A did not interfere with S's effectiveness. Apparently A in the doses given does not alter beta receptor response to agonists or antagonists.

This study demonstrates, in agreement with Gomoll and McKinney (19), the effectiveness for beta adrenoceptor blockade by S. However, the family of curves in this study were atypical, i.e., they had an ellipsoidal configuration with convergence at the 10.0 yg/kg dose of ISP and the 55 curves appeared to tail-off asymptotically at 24-32 percent blockade rather than approach zero blockade. Gomoll and McKinney (19) showed that, despite an 8.0 mg/kg dose of S (given alone), the heart rate response to supramaximal doses of ISP was not dulled. However, the response of heart rate to lesser amounts of ISP was attenuated, i.e., dose-response curves were shifted to the right by successively higher S doses . After autonomic blockade, two factors contribute to evoke a falsely elevated calculated percent chronotropic blockade in response to 10.0 yg/kg of ISP, thus creating the convergence of the family of curves. First, after autonomic blockade, the basal heart rate was elevated above control. Second, despite autonomic blockade with S and A, heart rate accelerated to nearly 300 beats per minute in response to a challenge of 10.0 yg/kg of ISP (Table 6). This heart rate approaches the heart rate achieved during maximal exertion (13, 24, 64) in which vagal withdrawal

(52, 53) and adrenergic efferent activity (52) are maximal. Because a physiologically limiting heart rate response occurred with 10.0 yg/kg ISP, a lesser heart rate change occurred during autonomic blockade than in the control (due to the lower control basal value) from this amount of ISP and a disparity in the calculated percent blockade resulted. Zero percent blockage could not be achieved 56 because a calculated value of unity could not be obtained in the ISP heart rate response ratio (AHR due to ISP after autonomic blockade/AHR due to ISP without autonomic block­ ade) , a part of the formula for calculating percent block­ ade. Consequently, a false value greater than zero block­ ade was calculated in response to 10.0 yg/kg of ISP. Higher ISP dosage probably would not produce a different calculated value and an asymptote would be expected at around 25 percent blockade.

Heart Rate-Contractility Relationship. A surprising, yet not totally unexpected result, was the lack of a relationship between heart rate and indices of left ven­ tricular contraction (dP/dt max and Vmax) in these con­ scious dogs. Such a relationship, when present, is termed

the Bowditch effect or chronotropic-inotropism. It is thought to arise from incomplete restitution of intra­ cellular calcium during the abbreviated diastole, the period during which calcium is pumped extracellularly or into sarcoplasmic reticulum. Calcium, therefore, accumu­ lates intracellularly with increasing heart rates (33), and serves as a positive inotrope. Studies demonstrating increased ventricular function resulting from increasing heart rates (12, 51, 65) have been conducted largely in dogs anesthetized with ganglioplegic drugs, i.e., sodium pentobarbital and, to a lesser extent, morphine-chloralose. 57

Other studies, performed on conscious dogs (21, 42, 43) have refuted the Bowditch effect and stated that it is an artifact of the anesthetic or surgical interventions. This study confirms the absence of a Bowditch effect in the con­ scious dog. Therefore, when an alteration in an index of contractility was observed in this study on S, such alter­ ations were probably not due to heart rate.

Sotalol-Atropine Sequences. S (8.0 mg/kg) and A

(0.2 mg/kg) given in reversed sequences and in combination allowed for separate examinations of sympathetic and para­ sympathetic contributions to chronotropy and inotropy. The results will be discussed as trends because a larger sample size was needed for the ANOVA to reveal changes of statisti­ cal significance. Means for heart rate, dP/dt max and Vmax during the control period for each experimental group were similar, therefore, comparisons of values between groups was permissible.

As shown in Table 8, when A was given to conscious dogs, heart rate accelerated S9%. When S was given before, after, or in combination with A, heart rate accelerated only 16-21% above control, and decreased 14% when given alone. Thus, this 16-21% cardioacceleration represents the acceleration produced by the vagolytic action of A; while the percent of cardioacceleration obtained without S represents both the vagolytic and sympathomimetic, or 58 direct sino-atrial nodal stimulating, effects of A (14). Furthermore, with respect to the chronotropic or ino­ tropic effect, the order with which the drugs were given was inconsequential. When S was given alone, both heart rate and dP/dt max decreased, but Vmax did not. Investigators previously have demonstrated S’s freedom from myocardial depressant effects when given alone (8, 9, 16, 19, 22, 48) or combined with A (6, 19). If it can be presumed, as has been claimed, that Vmax is determined only by the contractile state (10, 57, 59), then this study shows that S probably does not affect myocardial contractility. How, then, is the discrepancy between dP/dt max and Vmax reconciled? dP/dt max is known to have four determinants (39): heart rate, preload, after­ load, and contractile state; a reduction in any one of them could account for the reduction in dP/dt max. Left ventricular end-diastolic pressure (LVEDP), a measure of preload, which was estimated because the sealed manometer could not be referenced to atmosphere after im­ plantation, was not changed from control by any S-A combi­ nation. Others (8, 19, 34), using anesthetized dogs, also report that sotalol has no significant effect on LVEDP. Noble (43) reports that initial fiber length (preload) changes had no effect on dP/dt max in conscious dogs. 59

These evidences suggest that preload is unlikely the cause for the dP/dt reduction. Some investigators have proposed that the negative inotropism, observed after S, was due to the inotropic index’s dependence upon heart rate (8, 9, 16, 19). The results of this study contradict the rate-dependency pro­ posal for two reasons. First, no relationship existed be­ tween heart rate and dP/dt max, as shown earlier. Second, since the addition of A to S, in any sequence, induced a substantial heart rate increase from control (16-211) while dP/dt max remained substantially decreased below con­ trol (24-28%), heart rate could not have influenced dP/ dt max negatively under this condition. A more likely proposal, involving afterload, appears tenable for this study. Although arterial pressure (after­ load) was not monitored, hemodynamic data obtained by other investigators (19, 48) revealed that S reduces mean arterial pressure. Despite an elevated total peripheral vascular resistance in response to S, the paradoxical mean arterial pressure resulted from a rate-related reduction in cardiac output; stroke volume was either increased (19) or not af­ fected (48). Wallace et al. (65), Mason (39) and Wilden- thal et al. (66) showed that dP/dt max shifts in the same direction as alterations in afterload. It appears, therefore, that the reduced afterload, evoked indirectly 60 by S ’s depressant effect on heart rate, may have consti­ tuted the mechanism for the depressed dP/dt max in this s tudy. Brooks et al. (8) proposed another explanation of why dP/dt max decreased but Vmax did not. When dogs whose catecholamine stores were depleted by pretreatment with reserpine, were given S, neither dP/dt max nor Vmax de­ creased. This indicates that S does not have direct myo­ cardial depressant action, but only reduces the myocardial contractile state by interference with beta-adrenergic drive. That dP/dt max decreased but Vmax did not in normal

(non-reserpinized) dogs indicates that a small amount of beta-adrenergic drive exists in the resting dog, and con­ firms that dP/dt max and Vmax have different determinants. The same mechanism as described above by Brooks et al. may be operative after the vagolytic doses of A given in this study. A is thought to have no direct effect on the myocardium, even in massive doses (68), however, in higher doses than are needed to antagonize acetylcholine, A may block or reduce responses to norepinephrine (69). The response of dP/dt max to A was a slight reduction (5-8%) regardless if it was given alone or with S. If it can be presumed that a 0.2 mg/kg dose of A to conscious dogs is sufficient to inhibit norepinephrine responses, then A may have contributed to a slightly reduced dP/dt max. SUMMARY AND CONCLUSIONS

A series of studies designed to investigate the ino­ tropic and chronotropic effect of S (0.5-16.0 mg/kg) com­ bined with a vagolytic dose of A (0.2 mg/kg) was investi­ gated in conscious dogs. The studies included: (1) du­ ration of ISP response; (2) dose-response for ISP, A, and S; (3) effectiveness of beta-adrenoceptor blockade; (4) HR-contractility relationship; and (5) alternating S-A sequences. S given with A, produced effective beta-adrenergic blockade without myocardial depression. 2.0 mg/kg of S produced an 80% chronotropic blockade to an ISP dose

(0.3 mg/kg) which causes a 95 beat per minute HR increase from control. A four-fold increase (8.0 mg/kg) from the minimal effective dose of S (2.0 mg/kg) did not elicit a change in Vmax, but dP/dt max decreased. The alteration in dP/dt max presumably was related to S's known hemodynamic effect, viz., a reduced afterload secondary to a rate- dependent reduced cardiac output. Since Vmax is known to be not altered by ventricular loading conditions, it

61 62 appeared to accurately reflect S's lack of effect on the myocardial contractile state. An interaction between A, ISP and S evoked an atypically configured family of curves relating S dosage to percent chronotropic blockade to incremented ISP dosage. A, in the presence of a supramaximal ISP dose (10.0 pg/kg), created a condition which falsely elevated the calculated percent chronotropic blockade. Autonomic blockade, by S plus A, revealed an intrinsic heart rate which unexplainably was greater than the control basal rate. The intrinsic heart rate at low to moderate S doses (0.5-4.0 mg/kg) reduced with each succeeding increase in S dosage suggesting a direct depressive effect on the sino-atrial node. Another unexplained mechanism probably contributed also to the HR reduction after autonomic block­ ade since S given alone decreased HR only 19% as much as the same amount of S given after an atropine-induced vagolytic rate. Larger doses of S (8.0 and 16.0 mg/kg), however, demonstrated a decreased attenuation of heart rate reflecting a "rebound" chronotropic effect, and HR instability (16.0 mg/kg). These characteristics of large doses suggest S may possess intrinsic sympathomimetic activity. This finding is unique and may reflect an auto­ nomic reactivity difference in conscious subjects not seen in anesthetized subjects. 63

A relationship between HR and contractility (Bow­ ditch effect) could not be demonstrated in the conscious dogs of this study. This finding contradicts a tra­ ditional physiological phenomenon established under conditions other than consciousness. It may, therefore, again reflect an autonomic reactivity or cardiovascular control mechanism behavior difference between conscious dogs and anesthetized dogs. Alterations of contractility indices observed in this study, therefore, were probably not due to heart rate. With respect to the inotropic and chronotropic effect of a S + A combination, the sequence of drug administration was inconsequential. That HR accelerates 89% from control after A given alone, decreases 14% after S given alone, and accelerates only 16-21% after S + A in any sequence indi­ cates that A has a significant chronotropic sympathomimetic effect in addition to its vagolytic effect. A consistent but insignificant negative inotropic effect of a vagolytic dose of A was suggested when dP/dt max was slightly reduced (5-8%) regardless if A was given alone or combined with S. BIBLIOGRAPHY

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