Myocardial Effects of Propofol in Hamsters with Hypertrophic Cardiomyopathy 

Six normal Syrian hamsters (strain FIB) and nine cardiomyopathic Syrian hamsters (strain BIO 14.6) were used in the current study (Bio Breeders, Fitchburg, MA). In this strain, all animals of both sexes develop hypertrophic cardiomyopathy from the age of 6 weeks. Care of the animals conformed to the recommendations of the Helsinki Declaration, and the study was performed in accordance with the regulations of the official edict of the French Ministry of Agriculture. All animals were 6 months of age. Body weight (BW), heart weight (HW), and left ventricular weight (LVW) were determined at the moment of killing. HW/BW and LVW/BW ratios were calculated. Cardiac hypertrophy was determined by dividing the HW/BW value of each cardiomyopathic hamster by the mean HW/BW value in normal hamsters.

Twenty-two left ventricular papillary muscles (1–2 from each hamster) were studied. After brief anesthesia with ether, the hearts were quickly removed and left ventricular papillary muscles were carefully excised and suspended vertically in a 200-ml jacketted reservoir with Krebs-Henseleit bicarbonate buffer solution containing (mmol *symbol* l sup -1) NaCl 118, KCl 4.7, MgSO⁴1.2, KH²PO sub 4 1.1, NaHCO³25, CaCl²2.5, and glucose 4.5. The Krebs-Henseleit solution was daily prepared with highly purified water (Ecopure, Bioblock, Illkirch, France). The jacketted reservoir was maintained at 29 degrees Celsius with a thermostatic water circulator (Polystat 5HP, Bioblock) and a continuous monitoring of the solution temperature (Pt 100 temperature probe, Bioblock). Preparations were field-stimulated at 3/min by two platinum electrodes with rectangular wave pulses of 5-ms duration just above threshold. The bathing solution was bubbled with 95% Oxygen²/5% CO O²/5% CO², resulting in a pH of 7.40. After a 90-min stabilization period at the initial muscle length at the apex of the length-active isometric tension curve (L^max), papillary muscles recovered their optimal mechanical performance, which remained stable for many hours.

Control values of each mechanical parameter were recorded. Because propofol is insoluble in aqueous media, we tested the commercially available form of propofol in which a soya bean emulsion is the solvent (Diprivan, Zeneca Pharma, Cergy-Pontoise, France). The solvent has been shown to be devoid of significant effect on intrinsic myocardial contractility. [4] Concentrations of propofol during anesthesia range from 1 to 10 micro gram *symbol* ml -1. [1,12,13] Propofol is highly bound (96–98%) to plasma protein, but this does not seem to influence its rapid disappearance from the blood and extensive tissue distribution. [13] Thus, three concentrations of propofol were tested in a cumulative manner: 1 micro gram *symbol* ml sup -1 (5.6 micro mol *symbol* l sup -1). 3 micro gram *symbol* ml sup -1 (16.8 micro mol *symbol* l sup -1), and 10 micro gram *symbol* ml sup -1 (56.4 micro mol *symbol* l sup -1), with 15 min elapsing between each dose, as previously reported in rat myocardium. [4].

The electromagnetic lever system has been described previously. [14] Briefly, the load applied to the muscle was determined by means of a servomechanism-controlled current through the coil of an electromagnet. Muscular shortening induced a displacement of the lever, which modulated the light intensity of a photoelectric transducer. All analyses were made from digital records of force and length obtained with a computer, as previously described. [4] The recording speed was one A/D (12 bits) conversion every 1 ms, for a total recording time of 500 ms.

Conventional mechanical parameters at L^maxwere calculated from three twitches. The first twitch was isotonic and was loaded with the preload corresponding to L^max. The second twitch was abruptly clamped to zero-load just after the electrical stimulus; the muscle was released from preload to zero-load with a critical damping to slow the first and rapid shortening overshoot resulting from the recoil of series passive elastic components, as previously reported [15]; the maximum unloaded shortening velocity (V^max) was determined from this twitch. The third twitch was fully isometric at L sub max.

The mechanical parameters characterizing the contraction and relaxation phases, and the coupling between contraction and relaxation are defined as follows.

Contraction Phase. We determined V^maxusing the zero-load clamp technique; maximum extent of shortening (DL^max) and maximum shortening velocity (sub max Vc) of the twitch with preload only; maximum isometric active force normalized per cross-sectional area (AF); and the peak of the positive force derivative normalized per cross-sectional area (+dF *symbol* dt sup -1). V^maxand AF tested the inotropic state under low and high loads, respectively.

Relaxation Phase. We determined maximum lengthening velocity of the twitch with preload only (sub max Vr) and the peak of the negative force derivative at L^maxnormalized per cross-sectional area (-dF *symbol* dt sup -1). These two parameters tested the lusitropic state under low- and high-loading conditions, respectively. Because changes in the contraction phase induce coordinated changes in the relaxation phase, variations in contraction and relaxation must be considered simultaneously to quantify drug-induced changes in lusitropy. Therefore, indexes of contraction-relaxation coupling have been developed. [16].

Contraction-Relaxation Coupling. Coefficient R 1 =^maxVc/sub max Vr tests the coupling between contraction and relaxation under low load. Under isotonic conditions, the amplitude of sarcomere shortening is twice that observed under isometric conditions. [17] Because of the lower sensitivity of myofilament for calcium when cardiac muscle is markedly shortened under low load, relaxation proceeds more rapidly than contraction, apparently due to the rapid uptake of calcium by the sarcoplasmic reticulum (SR). Thus, R1 (contraction-relaxation coupling under low load) is significantly less than 1 and tests SR function. Coefficient R2 =(+dF *symbol* dt sup -1/-dF *symbol* dt sup -1) tests the coupling between contraction and relaxation under high load. When the muscle contracts isometrically, sarcomeres shorten very little. [17] Because of a higher sensitivity of myofilament for calcium, [18] the relaxation time course is determined by calcium unbinding from troponin C rather than by calcium sequestration by the SR. Thus, R2 (contraction-relaxation coupling under heavy load) is greater than 1 and reflects myofilament calcium sensitivity.

At the end of the study, the muscle cross-sectional area was calculated from the length and weight of papillary muscle, assuming a density of 1. Shortening and lengthening velocities were expressed in L sub max *symbol* S sup -1, force in mN *symbol* mm sup -2, and force derivative in mN *symbol* mm sup -2 *symbol* s sup -1.

Data are expressed as mean plus/minus SD. Control values in normal hamsters and those with cardiomyopathy were compared by means of the Student's t test. The effects of propofol in normal hamsters and those with cardiomyopathy were compared by repeated-measures analysis of variance and Student-Newman-Keuls test. All P values were two-tailed, and a P value of less than 0.05 was required to reject the null hypothesis. Statistical analysis was performed on a personal computer using PCSM software (Deltasoft, Meylan, France).

HW and LVW were not significantly different between normal hamsters and those with cardiomyopathy. However, the HW/BW and LVW/BW were significantly greater in cardiomyopathic hamsters, indicating marked cardiac hypertrophy (Table 1). The intrinsic mechanical performance of papillary muscles from hamsters with cardiomyopathy was significantly lower during isometric (AF, +dF *symbol* dt sup -1) and the isotonic (DL^max, V^max,^maxVc) twitches (Table 2). R1, which tests the contraction-relaxation coupling under low load, and thus SR function, was not significantly different between the two groups. In contrast, R2, which tests the contraction-relaxation coupling under high load, and thus the myofilament calcium sensitivity, was significantly lower in cardiomyopathic hamsters than in controls.

Propofol induced no significant inotropic effect on normal muscles as shown by the absence of changes in V^max(Figure 1) and active isometric force (Figure 2). No differences in these inotropic parameters were observed between normal hamsters and those with cardiomyopathy (Figure 1and Figure 2), but a slight significant increase in V^maxwas noted at 3 micro gram *symbol* ml sup -1 (+4 plus/minus 6%) and 10 micro gram *symbol* ml sup -1 (+7 plus/minus 6%) propofol in cardiomyopathic muscles (Figure 1). Because contractile properties varies from one papillary muscle to another in cardiomyopathic hamsters, papillary muscles were divided into two groups: those with a severe myocardial failure (i.e., AF of less than 25 mN *symbol* mm²; n = 6; AF = 16 plus/minus 8 mN *symbol* mm²) and those with a moderate myocardial failure (n = 7, AF = 32 plus/minus 6 mN *symbol* mm²). At the highest concentration of propofol (10 micro gram *symbol* ml sup -1), there was no significant difference in the percent change in AF between papillary muscles with severe myocardial failure (113 plus/minus 8%) and those with moderate myocardial failure (102 plus/minus 10%).

Under isotonic conditions, propofol had no significant lusitropic effect (sub max Vr, data not shown) in normal hamsters, resulting in no significant changes in contraction-relaxation coupling (R1), and no significant differences were noted between normal hamsters and those with cardiomyopathy (Figure 3). Under isometric conditions, propofol had no lusitropic effect (-dF *symbol* dt sup -1, data not shown) in normal hamsters, resulting in no significant changes in contraction-relaxation coupling (R2), and no significant differences were noted between normal hamsters and those with cardiomyopathy (Figure 4).

In the present study, we observed that propofol induced no significant effects on the intrinsic mechanical properties of left ventricular papillary muscles from normal hamsters and that there were no significant differences in the effects of propofol in normal hamsters and those with cardiomyopathy.

Numerous studies indicate that propofol administration is associated with cardiovascular depression. [19,20]. However, the precise effects of propofol on myocardial contractility remain debatable. The effects of a drug on intrinsic myocardial contractility are difficult to assess in vivo because of changes in heart rate, preload, and afterload, [21] especially with anesthetic agents known also to decrease oxygen demand and central nervous system activity, and consequently cardiac output. Moreover, propofol is known to markedly decrease preload [2] and sympathetic activity. [1] Most recent in vitro studies observed that propofol has no significant inotropic effect on myocardium in various species, including the rat, rabbit, and dog. [4–7] Nevertheless, it is clear that a negative inotropic effect can be observed with propofol in the ferret [22] and guinea pig myocardium, [5] and in certain experimental conditions that are associated mainly with ischemia. [4,6,23] The mechanisms of the negative inotropic effect of propofol in the guinea pig and the ferret myocardium have been explained. Propofol appears to decrease calcium availability within the myocardium, based upon studies in ferrets in which there is no change in the myofilament calcium sensitivity. [22] Depression of the cardiac action potential plateau and duration in guinea pig myocardium [5] suggest that calcium current may be depressed, but this effect does not occur in rat myocardium. [5] However, it should be pointed out that Azuma et al. [5] tested supratherapeutic propofol concentrations (i.e.,up to 600 mol *symbol* 1 sup 1) in guinea pig myocardium, and that Park and Lynch [24] were unable to demonstrate depression of action potential duration and rate of depolarization at lower concentrations in the guinea pig, suggesting that the effect of propofol on calcium cardiac currents is modest. Our study showed that propofol induced no significant effect on intrinsic myocardial contractility in the hamster, as previously reported in rat, rabbit, and dog myocardium. [4–7]

Propofol did not modify contraction-relaxation coupling under low load (R1). Under isotonic conditions, the amplitude of sarcomere shortening is twice that observed in isometric conditions, [17] and the time course of isotonic relaxation occurs earlier and more rapidly than that of isometric relaxation, partly through two mechanisms [18]:(1) the easier removal of calcium from troponin C, due to a decrease in myofilament calcium sensitivity, and (2) the rapid uptake of calcium by the SR. Under low load, the SR appears to play a major role in the regulation of isotonic relaxation. Our results therefore suggest that propofol did not modify the uptake of calcium by the SR. This result contrasts with that previously obtained in rat myocardium. [4] Indeed, in rat myocardium, propofol increased R1, suggesting a decrease in the uptake SR function. [4] Two hypotheses can explain this discrepancy:(1) the effects of propofol on SR function were too small in hamster myocardium to be detected; indeed, the effects of propofol in rat myocardium were moderate because it did not induce any significant negative inotropic effect [4];(2) the effects of propofol on SR function did not occur in hamster myocardium; it is well known that the contribution of SR function to contractility is higher in the rat than in other species. [25].

The contraction-relaxation coupling under high load (R2) was not modified by propofol. Under isometric conditions and because of the slight sarcomere shortening, myofilament calcium sensitivity is less decreased than in isotonic conditions and becomes the limiting step that appears to play a major role in the regulation of the time course of isometric relaxation. The absence of any lusitropic effect of propofol under high load suggests that it did not modify the myofilament calcium sensitivity, as previously reported in rat myocardium. [4]

Genetically induced cardiomyopathy in Syrian hamsters is characterized by the progressive occurrence of focal myocardial degeneration, fibrosis, and calcifications during the life of the animal. [10,11] At age 30–40 days, histologic lesions become apparent and myocardial performance decreases. Further cardiac changes include hypertrophy and/or dilation, depending on the strain, then congestive heart failure and death. In our study, myocardial contractility was markedly impaired in cardiomyopathic muscles, as reflected by the decrease in V^maxand AF (Table 2). The decreased myocardial contractility in cardiomyopathic hamsters may be explained by the decreased activity of G proteins, [26] decreased sarcolemmal Calcium sup ++ and Sodium sup +-Potassium sup + ATPase activities, [27] alterations in Sodium-Calcium exchange, [28] decreased conductance [28] and density [29] of voltage-sensitive calcium channels, alterations in the creatine kinase system, [30] modifications in myofilament calcium sensitivity, [30] alteration in actin regulatory proteins, [31] and isomyosin shift. [32] Nevertheless, a decrease in SR function is not observed in all strains, and the strain used in the current study (BIO 14.6), which develops hypertrophic cardiomyopathy, has been shown to exhibit normal SR function. [33] In the current study, the lack of significant increase in R1 is consistent with these results and concords with results previously obtained in another strain with hypertrophic cardiomyopathy (UMX 7.1). [34] In contrast, R2 of hamsters with cardiomyopathy was lower than that of the controls, suggesting that myofilament calcium sensitivity was lower in hamsters with cardiomyopathy, as previously reported. [8,9,31].

Because of the complex action of anesthetic agents on cardiac muscle and because of the various pathologic changes observed in the myocardium of cardiomyopathic hamsters, it is not easy to predict the mechanical effects of anesthetic agents on this diseased myocardium. Indeed, it has been demonstrated that the mechanical effects of ketamine [8] and etomidate [9] differ in normal and hamsters with cardiomyopathy. In contrast, in the current study, we observed that the mechanical effects of propofol did not differ in left ventricular papillary muscles from normal and cardiomyopathic hamsters. Although the effect of propofol on V^maxwas not significantly different in both groups, a slight but significant increase in V^maxwas noted in cardiomyopathic hamsters. This slight effect must be interpreted with caution. Three hypotheses may explain this slight increase in V^max:(1) propofol actually increased V^max;(2) the increase in V^maxwas related to a slight increase in V^maxwith time in cardiomyopathic hamsters despite a prolonged recovery period before drug testing; it should be pointed out that such a slight increase in V^maxwas also previously noted with etomidate in hamsters with cardiomyopathy [9];(3) the increase in V^maxwas related to the solvent, which may have served as a metabolic substrate in cardiomyopathic muscle, as previously suggested. [22] In any event, this very moderate increase in V^maxwithout significant changes in active isometric force has probably no clinical relevance. Because hamsters with cardiomyopathy were relatively young (age 6 months) and because myocardial abnormalities are more prominent in end-stage disease, it is possible that the result would have been different had the animals been older and sicker. However, myocardial abnormalities are not homogeneous among different hamsters of the same age and are not homogeneous within the entire myocardium of each hamster. We observed several deaths in 6-month-old hamsters, and thus, when studying older hamsters with cardiomyopathy, one cannot rule out the hypothesis that these older hamsters had less severe cardiomyopathy. It is reason why we divided papillary muscles into two groups, those with severe myocardial failure and those with moderate myocardial failure, and we observed that the effect of propofol did not significantly differ between these two groups.

The following points must be considered in the assessment of the clinical relevance of our results. First, because this study was conducted in vitro, it dealt only with intrinsic myocardial contractility. Observed changes in cardiac function after in vivo propofol administration also depend on modifications in venous return, afterload, and compensatory mechanisms. This point should be of special importance in patients with cardiomyopathy whose cardiac function does not depend only on intrinsic contractility but also on preload, afterload, and sympathetic activity.* Second, this study was conducted at 29 degrees Celsius at a low-stimulation frequency; however, papillary muscles must be studied at this temperature because stability of mechanical parameters is not sufficient at 37 degrees Celsius and at a low frequency because high-stimulation frequency induces core hypoxia. [35] Moreover, higher stimulation rates frequently induce extrasystoles in cardiomyopathic muscles, resulting in an unreliable recording of mechanical parameters. Third, it was performed on hamster myocardium, which differs from human myocardium. In hamster myocardium as in rat, a negative staircase effect is observed (an increase in stimulation frequency decreases force), contractility is high, and myosin isoforms are predominantly of the V1 type. Furthermore, because propofol is highly bound to plasma protein and because the bathing solution was protein-free, the concentrations tested might be considered high.

In conclusion, in studies conducted on isolated left ventricular papillary muscle, propofol did not modify intrinsic myocardial contractility in normal hamsters, and no significant differences were observed between normal hamsters and those with cardiomyopathy. These results could be important because most anesthetic agents decrease myocardial contractility [36] and propofol is widely used for the sedation of critically ill patients whose cardiac function may be impaired. However, the indirect effects of propofol may be more important than its direct cardiac effects in such conditions.

*Diedericks J, Foex P, Sear JW, Ryder WA: Haemodynamic effects of propofol infusion: Dose-response study in compromised canine hearts (abstract). Br J Anaesth 64:393P, 1990.