Myocardial Effects of Halothane and Isoflurane in Senescent Rats

Adult (3-month-old) and senescent (24-month-old) male Wistar rats from the same origin (Iffa Credo, Lyon, France) were studied. Care of the animals conformed to the recommendations of the Helsinski Declaration, and the study was performed in accordance with the regulations of the official edict of the French Ministry of Agriculture. Body weight was determined at the moment of killing; heart weight, and left ventricular weight were determined after. Heart weight to body weight and left ventricular weight to body weight ratios were calculated.

Left ventricular papillary muscles were studied in a Krebs-Henseleit bicarbonate buffer solution (118 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO⁴, 1.1 mm KH²PO⁴, 25 mm NaHCO³, 2.5 mm CaCl², and 4.5 mm glucose) maintained at 29°C with a thermostatic water circulator. Muscles were field stimulated at 12 pulses/min with rectangular wave pulses lasting 5 ms just above threshold. The bathing solution was bubbled with 95% oxygen and 5% carbon dioxide, resulting in a pH of 7.40. After a 60 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. 2,6In a preliminary study, we verified that the stability of the preparation was obtained over 120 min in papillary muscles from adult (n = 7) and senescent (n = 6) rats (data not shown). The extracellular calcium concentration ([Ca²⁺]⁰) was decreased from 2.5 to 1 mm because rat myocardial contractility is nearly maximum at 2.5 mm, and hence it is difficult to quantify inotropic changes without previously decreasing [Ca²⁺]^0.We performed additional experiments at a [Ca²⁺]⁰of 0.5 mm, because the lusitropic effects of halothane are modulated by [Ca²⁺]⁰, and postrest potentiation is more sensitive at low [Ca²⁺]⁰. 13 

Halothane (Fluotec 3; Cyprane Ltd, Keighley, UK) and isoflurane (Fortec 3, Cyprane Ltd) were added to the carbon dioxide–oxygen mixture with calibrated vaporizers. 2,6Anesthetic concentrations in the gas phase were monitored continuously using a infrared analyzer (Artema MM 206SD; Taema, Antony, France). Halothane concentrations ranged from 0.3 to 1.5 vol%, and those of isoflurane from 0.4 to 2 vol%, equivalent to 0.5 to 2.5 minimum alveolar concentration (MAC), respectively. 14Since MAC values of halogenated anesthetics are lower in senescent rats, 15we also compared halogenated anesthetics at equipotent concentrations (expressed as MAC). A correction factor of −17% was applied to estimate real MAC values in senescent rats. 15A 20-min equilibration period was allowed between each anesthetic concentration.

The electromagnetic lever system has been described previously. 13All analyses were made from digital records of force and length obtained with a computer. 13Conventional mechanical variables 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 fully isometric at L^max. The third twitch was abruptly clamped to zero-load just after the electrical stimulus; the muscle was released from preload to zero-load with critical damping. 13We determined V^maxusing the zero-load clamp technique and maximum shortening velocity (^maxVc) of the twitch with preload only, maximum isometric active force normalized per cross-sectional area (AF), and peak of the positive force derivative normalized per cross-sectional area (+dF/dt). We determined maximum lengthening velocity of the twitch with preload only (^maxVr) and peak of the negative force derivative at L^maxnormalized per cross-sectional area (-dF/dt). Because changes in the contraction phase induce coordinated changes in the relaxation phase, indexes of contraction–relaxation coupling thus have been developed to study lusitropy. 16R1 coefficient =^maxVc/^maxVr studied the coupling between contraction and relaxation under low load, and thus lusitropy, in a manner that is independent of inotropic changes. R1 tests SR uptake function. 2,13,16R2 coefficient = (+dF/dt)/(−dF/dt) studied the coupling between contraction and relaxation under high load, and thus lusitropy, in a manner that is less dependent on inotropic changes. R2 indirectly reflects myofilament calcium sensitivity. 2,13,16 

During rest in the rat myocardium, SR accumulates calcium and the first beat after the rest interval (B1) is more forceful than the last beat before the rest interval (B0). 13During postrest recovery, the SR-dependent part of activator calcium decreases somewhat toward a steady state, which is achieved in a few beats. AF during postrest recovery was studied at a [Ca²⁺]⁰of 0.5 mm, after a 1-min rest duration, and the rate constant of the exponential decay of AF (τ) was determined. 13τ is assumed to represent the time required for the SR to reset Ca²⁺homeostasis and thus was used to test SR function. 13At the end of the study, the muscle cross-sectional area was calculated from its length and weight, assuming a density of 1.

Total RNA was isolated from the individual left ventricle. 17cDNA probes were labeled by random priming or nick translation with an α[³²P] dCTP and the synthetic oligonucleotides with T4 polynucleotide kinase and γ[³²P] ATP. For Northern Blot analysis, 10 μg of total RNA were submitted to electrophoresis and then transfer was performed on Nylon Hybond-N membrane (Amersham; Les Ulis, France). For Slot Blot analysis, 1, 2, 4, and 8 μg of total RNA were directly applied to the membrane (Nylon Hybond-N membrane; Amersham). After ultraviolet irradiations to covalently link the RNA samples, the RNA blots were hybridized sequentially with an α-myosin heavy chain (MHC) oligonucleotide probe, 9a β-MHC cDNA probe, 9a SR Ca²⁺–ATPase cDNA probe, 18a 18S rRNA oligonucleotide probe, 9,18and a 25- to 30-mer oligo(dT) probe. 9,18The last two probes were used to quantitate 18S rRNA and total poly(A)⁺mRNA, respectively, in a given left ventricle and to normalize the measurements. 9,18Prehybridization, hybridization, and washing conditions were performed, as previously reported. 9,18The washed blots were exposed to x-ray films (Hyperfilm; Amersham) with Quanta III intensifying screens for 16–48 h at −70°C. The relative level of each mRNA species was determined in slot blots by scanning densitometry within the linear response range of the x-ray films. The densitometric scores of each specific mRNA were normalized to that of 18S rRNA or poly(A)⁺mRNA measured by oligo(dT).

Data are expressed as mean ± SD. Baseline values between groups were compared using the Student t  test. Comparison of several means was performed using a repeated-measures analysis of variance and the Newman–Keuls test (concentrations of anesthetic agents expressed as vol%) or multivariate analysis of variance (concentrations of anesthetic agents expressed as multiples of MAC). All probability values were two-tailed, and P  values less than 0.05 were required to reject the null hypothesis.

Body weight (490 ± 118 vs.  328 ± 38 g, P < 0.05), heart weight (1060 ± 180 vs.  710 ± 110 mg, P < 0.05), and left ventricular weight (760 ± 150 vs.  520 ± 90 mg, P < 0.05) were significantly higher in senescent than in adult rats. The heart weight to body weight ratio (2.23 ± 0.55 vs.  2.10 ± 0.38 mg/g, NS) and the left ventricular weight to body weight ratio (1.58 ± 0.33 vs.  1.58 ± 0.20 mg/g, NS) were not significantly different between senescent and adult rats.

The L^maxwas not significantly different between papillary muscles from senescent and adult rats. On the other hand, CSA and RF/TF were higher in the senescent group (table 1). The intrinsic mechanical inotropic performance of papillary muscles from senescent rats was significantly lower in isometric (AF, +dF/dt) and isotonic (V^max, ^maxVc, ΔL) conditions (table 1). Relaxation velocities were lower in senescent rats in isometric (-dF/dt) and isotonic (^maxVr) conditions (table 1). R1 was not significantly different between senescent and adult groups. In contrast, R2 was lower in senescent group (table 1).

Compared with the adult group, α-MHC mRNA concentration was lower in the senescent group whereas β-MHC mRNA concentration was higher (fig. 1). In addition, SR Ca²⁺–ATPase mRNA relative concentration was lower in the senescent group (fig. 1), even when these mRNA species were normalized to rRNAs (data not shown).

At [Ca²⁺]⁰of 0.5 or 1.0 mm, halothane induced a marked concentration-dependent negative inotropic effect in adult and senescent rats, as shown by the decreases in AF (fig. 2). The negative inotropic effect was less pronounced in senescent rats only in isotonic conditions at [Ca²⁺]⁰of 0.5 mm (V^maxat 1.5%: 63 ± 22% of baseline in senescent rats vs.  48 ± 15% of baseline in adult rats, P < 0.01) and in both isotonic and isometric conditions at [Ca²⁺]⁰of 1 mm (fig. 2). At a [Ca²⁺]⁰of 0.5 mm, isoflurane induced a moderate but significant negative inotropic effect, as shown by the decrease in AF (fig. 3) without significant differences between adult and senescent groups. At a [Ca²⁺]⁰of 1 mm, isoflurane induced a moderate negative inotropic effect only in isometric conditions (AF), without significant difference between adult and senescent rats (fig. 3).

Since MAC of halogenated anesthetic agents is 17% lower in senescent rats, we also plotted the inotropic effect as a function of MAC values in each group. At equipotent anesthetic concentrations, the negative inotropic effects of halothane remained significantly less pronounced in senescent rats compared with adult rats (fig. 4). In contrast, there was no significant difference between adult and senescent rats with isoflurane (fig. 4).

At a [Ca²⁺]⁰of 1.0 mm and under isotonic conditions, halothane induced a complex effect in adult rats: a positive lusitropic effect at low concentrations and a negative lusitropic effect at high concentrations (fig. 5). These effects significantly differed in senescent rats since halothane induced only a moderate negative lusitropic effect at high concentrations (fig. 5). At a [Ca²⁺]⁰of 0.5 mm and in isotonic conditions, halothane induced a marked negative lusitropic effect (increase in R1) in adult rats, which was significant at 2.0 vol% (fig. 5). On the other hand, in isotonic conditions, halothane induced no significant lusitropic effect in senescent rats (fig. 5).

Under isometric conditions, whatever was the [Ca²⁺]⁰concentration, halothane induced a moderate but significant negative lusitropic effect (decrease in R2) similar in adult and senescent rats (data not shown). For example, at a [Ca²⁺]⁰of 1 mm, the decrease in R2 induced by halothane (1.5%) was 79 ± 8% of baseline in the senescent group and 84 ± 9% of baseline in the adult group.

At a [Ca²⁺]⁰of 0.5 mm under low load, isoflurane induced a negative lusitropic effect at high concentrations similar in each group of rats (R1 at 2.0%: 112 ± 2% of baseline in the senescent group vs.  108 ± 22% in the adult group), whereas it induced no significant effect at a [Ca²⁺]⁰of 1 mm (data not shown). Under isometric conditions and whatever the [Ca²⁺]⁰, isoflurane induced no significant lusitropic effect in both groups (data not shown).

Isoflurane, but not halothane, slightly improved the postrest potentiation in both adult and senescent rats (table 2). There was no significant difference between the two groups of rats. Whatever the group studied, halothane and isoflurane did not significantly modify τ (table 2).

In the present study, we showed that despite marked alterations in myocardial contraction and relaxation in senescent myocardium, associated with reexpression of β-MHC mRNAs and a decrease in SR Ca²⁺–ATPase mRNA concentrations, halothane induced less pronounced negative inotropic and lusitropic effects in senescent than in adult rats, while isoflurane induced similar effects in both groups.

The mortality rate is 50% in 24-month old rats, approximately corresponding to humans aged 75 yr, both in terms of mortality and molecular and cellular alterations. 19We observed important alterations in the myocardium of these senescent rats. The rat ventricle contains two isoforms of myosin heavy chains, α and β isoforms within a high and slow ATPase activity, respectively. 20During aging, our results showed an inactivation of the gene coding for αMHC associated with a reexpression of the βMHC gene. This isomyosin shift results in a decreased ATPase activity which correlates with the decline in V^max, as previously reported. 9Several hypothesis can explain the decrease in force in senescent rats: (1) an important fibrosis, responsible for a decrease in the myofibrillar content 9; (2) actomyosin cross-bridge kinetic alterations, but this hypothesis remains to be tested. In contrast, several studies found no significant change in myofilament calcium sensitivity in the senescent rat. 21,22As shown by the increase in R1, the relaxation velocity is decreased in senescent papillary muscles. 9Calcium extrusion by Na⁺–Ca²⁺exchanger and uptake by the SR Ca²⁺–ATPase are the two major determinants of relaxation. However, in the rat, the SR plays a more important role in the relaxation process than in other mammalian species. We and others 9,23have observed a significant decrease in SR Ca²⁺–ATPase mRNA concentrations and Heyliger et al.  24have reported a reduction in the activity of the Na⁺–Ca²⁺exchanger during aging.

The cardiac effects of halothane and isoflurane have been widely studied in vitro . 1–6Vivien et al.  6showed that their negative inotropic effects were more pronounced in cardiomyopathic hamsters. Our study is the first to assess the effects of volatile anesthetics in senescent myocardium. Volatile anesthetics induce myocardial depression via  marked alterations in calcium homeostasis: (1) a decrease in the inward calcium current (I^Ca), related to an inhibition of both L-type calcium channels and Na⁺–Ca²⁺exchanger 25; (2) a decrease in SR function, which occurs to a lesser extent with isoflurane than with halothane 26; furthermore, halothane, but not isoflurane, increases calcium permeability of SR membrane, which contributes to depleting the SR calcium stores 26; this effect may be related to the fact that halothane, but not isoflurane, induces an increase in the opening duration of the cardiac SR calcium release channel (i.e. , ryanodine receptor) without altering the conductance or the opening likelihood 27; and (3) a decrease in Ca²⁺myofilament sensitivity, although the magnitude of this effect is still debated. 3In our study, halothane induced a lower negative inotropic effect in senescent than in adult rats, in spite of the marked contractile proteins alterations observed during senescence. During aging, ryanodine receptor density and activity are decreased 28,29without changes in mRNA concentrations (posttranscriptional regulation). 18On the other hand, in hypertrophied senescent myocytes, Walker et al.  30observed a proportional increase in the number of Ca²⁺channels while the action potential duration is prolonged in relation with a slowed inactivation of L-type Ca²⁺channel current, and consequently, the Ca²⁺current density is maintained. These differences may, at least partly, explain the lower negative inotropic effect of halothane in senescent rat.

In our study, isoflurane induced lower negative inotropic effects than halothane, in both groups, as previously reported. 2However, the inotropic effects of isoflurane were not significantly different between adult and senescent rats. The inotropic effects of isoflurane are mainly related to its effects on Ca²⁺channel and myofilament Ca²⁺sensitivity, without any significant effect on SR. The difference observed between halothane and isoflurane may be related to the absence of effect of isoflurane on the SR, or to the weak negative inotropic effect of isoflurane, which might have precluded us to show a difference during aging.

The MAC of halothane is decreased in senescent rats. 15The expression of AF as a function of MAC in each group confirmed that the negative inotropic effect of halothane is lower in senescent rats and also confirmed the lack of significant difference observed with isoflurane. As previously discussed in diseased myocardium, 6because little information is available about anesthetic potency during senescence in humans, we believe that it was important to report our results from a pharmacological (fig. 2) and a clinically relevant (fig. 4) point of view.

We observed that the lusitropic effect of halothane on R1 differs between adult and senescent papillary muscles. At a [Ca²⁺]⁰of 0.5 mm, halothane induced a negative lusitropic effect observed only in adult rats. This result is consistent with previous studies in adult rats, 2suggesting a decrease in SR calcium uptake. Halothane induces an alteration of cytosolic calcium concentration return during the relaxation phase, 31resulting from an inhibition of the SR Ca²⁺–ATPase. 32During senescence the activity of Ca²⁺–ATPase is reduced. 10Nevertheless, even if capture and extrusion lastings are increased, 10the amount of calcium pumped by the SR is unchanged in senescent rats. Moreover, the halothane-induced inhibition of SR Ca²⁺–ATPase is enhanced when phospholamban is phosphorylated, 32and Xu et al.  33have reported a significant age-related decrease in the phosphorylation of phospholamban by the endogenous SR-associated calmoduline kinase, which may be attributed to the decrease in the amount of δ-calmoduline kinase II, the predominant isoform present in cardiac cytosol and SR. These important senescence-induced differences may explain the lower negative lusitropic effects of halothane observed in senescent rats. In addition, we also demonstrated that the lusitropic effect of halothane on R1 depends on the Ca²⁺concentration. Using cardiac SR vesicles, Karon et al.  32have also reported that the halothane-induced inhibition of Ca²⁺uptake was more pronounced at low than at high Ca²⁺concentrations. These results can explain, at least partly, the different effects of halothane on R1 at 0.5 and 1.0 mm of calcium. However, at a high Ca²⁺concentration, no significant difference in the lusitropic effects of halothane was noted between adult and senescent rat. Isoflurane did not induce any significant lusitropic effect on R1 in senescent and adult rats, as previously reported. 2These results concord with the absence of reported effect of isoflurane on SR.

We showed that isoflurane, but not halothane, slightly improved postrest potentiation in both adult and senescent rats. Postrest potentiation provides a useful tool for examining complex underlying cellular processes, such as SR calcium release in cardiac muscle. 2,13In both groups, halothane and isoflurane did not affect postrest recovery, assessed by the rate constant, τ, of the exponential decay of force. As previously reported, in the adult rat, 2these results suggest that halogenated anesthetics do not significantly alter the recirculation fraction of calcium within the SR whatever the age of the animals.

Coefficient R2 tests the lusitropic state under isometric conditions and thus reflects myofilament Ca²⁺sensitivity. 2,16Several studies have reported that myofilament calcium sensitivity is not significantly modified during senescence. 21,22This is in accordance with data from our laboratory, showing that the active force-Ca²⁺relationship is similar between adult and senescent rats (S. Rozenberg, personal communication, 2002). Our results showed that halothane, but not isoflurane, induced a small decrease in R2 whatever the Ca²⁺concentration in both adult and senescent rats, but this effect has been previously related to the decrease in force and not to a decrease in myofilament Ca²⁺sensitivity. 6,34These results suggest that the difference in the myocardial effects of halogenated anesthetics do not rely on different action on myofilament Ca²⁺sensitivity.

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 addressed only intrinsic myocardial contractility. Observed changes in cardiac function after anesthetic administration also depend on modifications in heart rate, venous return, afterload, sympathetic nervous system activity, and compensatory mechanisms. Second, this study was carried out at 29°C and at low-stimulation frequency; however, papillary muscles must be studied at this temperature because the stability of mechanical parameters is not sufficient at 37°C and at low frequency because high-stimulation frequency may induce core hypoxia. 2,13Third, we studied 24-month-old Wistar rats whose ventricular function is not altered, 18and this is not the case in very old rats (>28 month old). 35Further studies may be useful in older animals. Fourth, it was performed on rat myocardium, which differs from human myocardium, but the effects of volatile anesthetics on the myocardium appear to be very similar among species, at least for halothane and isoflurane. 36 

In conclusion, in spite of marked alterations in the senescent myocardium, the inotropic and lusitropic effects of halothane were less important in senescent than in adult rats, whereas the effects of isoflurane were similar. These differences (adult vs.  senescent rats and halothane vs.  isoflurane) are probably related to differences in SR function and in the halogenated anesthetics-induced effects on the SR.

The authors thank Anne Marie Lompré, Ph.D., (INSERM IFR 75, Signalisation et Innovation Thérapeutique, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Sud, Chatenay-Malabry, France) for the gift of SR Ca²⁺–ATPase cDNA and βMHC cDNA, Claude Sebban, Ph.D., and Brigitte Tesolin-Decros, Ph.D., (Laboratoire de Biologie du Vieillissement, Hôpital Charles Foix, Ivry sur Seine, France) for providing senescent rats, and David Baker, M.D., F.R.C.A. (Department of Anesthesiology, CHU Necker-Enfants-Malades, Paris, France) for reviewing the manuscript.