Effects of Isoflurane and Sevoflurane on Intracellular Calcium and Contractility in Pressure-overload Hypertrophy

All experimental procedures were reviewed and approved by the Animal Care and Use Committee of the Mayo Foundation. Protocols were completed in accordance with National Institutes of Health guidelines and in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council). Right ventricular hypertrophy was induced by the method of pulmonary artery banding in the cat first reported by Spann et al.29,30and adapted to ferrets by Gwathmey and Morgan.31,32Ferrets that developed ascites or pleural effusion were considered to have developed decompensated right heart failure and were excluded from the analysis. Ferret ventricle was chosen because the level of function of the main proteins involved in [Ca²⁺]ⁱhomeostasis in the heart (sarcoplasmic reticulum Ca-ATPase, SERCA; Na-Ca exchanger, NCX; and plasma membrane Ca-ATPase, PMCA) is similar to human ventricle.33,34 

Ferrets were anesthetized 1–2 months after operation with halothane in 100% oxygen and the hearts rapidly excised and placed in oxygenated physiologic salt solution. Hearts were removed and perfused briefly with physiologic salt solution while appropriate papillary muscles (cross sectional area ≤1.2 mm²) were excised from the right ventricle. To isolate single ventricular myocytes, the right ventricular free wall was removed en bloc  and processed according to the previously published method.28 

Control experiments established that sham surgery (banding the pulmonary artery without constriction) did not alter the contractile response of isolated papillary muscles to either volatile anesthetic when compared to nonoperated hearts. Therefore papillary muscles from nonoperated normal animals was used for comparison.31 

Aequorin (Friday Harbor Laboratories, Friday Harbor, WA) was introduced into superficial cells of the papillary muscles using published methods.16,35The light emitted by aequorin was detected by a photomultiplier as previously published.16Digital signal averaging was used to obtain a satisfactory signal-to-noise ratio. Light measurements were converted into units of fractional luminescence (measured intensity/peak light intensity) by dividing them by the peak light intensity recorded under the optical conditions of the experiment when all the remaining aequorin was exposed to a saturating concentration of calcium.36To obtain peak light intensity, the cell membranes of the preparation were lysed with 4% Triton X-100 (Sigma; St Louis, MO) and the light emitted was integrated electronically.35Peak light intensity was corrected for consumption of aequorin by subtracting the amount of light emitted during each interval.37The intracellular [Ca²⁺] was determined from the fractional luminescence by referring to an in vitro  calibration curve.35–37 

Single isolated myocytes were loaded for 2–3 min with the acetoxymethyl ester of the fluorescent Ca²⁺indicator fluo-3 (Molecular Probes, Eugene, OR) at an extracellular concentration of 1 μm.28Fluo-3 was chosen for the intracellular Ca²⁺indicator because it undergoes an approximately 100-fold increase in fluorescence upon Ca²⁺binding. This property makes it particularly well suited for measuring Ca²⁺transients because it improves the signal-to-noise ratio. The rate and extent of shortening of fluo-3 loaded cells was not different from unloaded controls. A comparison of fura-2 and fluo-3 in pilot studies indicated that the two indicators yielded similar results and that the fluo-3 signals were less noisy.

Cells were prepared according to the previously published method and electrically stimulated by a pair of platinum electrodes to reach a steady-state level of intracellular Ca²⁺.28The electrodes carried square-wave pulses of 5 ms duration, at a voltage 10% above threshold, and at a rate of 0.25 Hz. All experiments were carried out at 23°C. At this temperature the extrusion of fluo-3 from ferret ventricular myocytes is minimal compared with that occuring at 37°C²⁸. Fluo-3 fluorescence was measured by a fluorometer (C&L Instruments, Hummelstown, PA) mounted on an inverted microscope (TE300; Nikon, Melville, NY). The fluo-3 was excited with 485 nm light from a xenon bulb and Ca²⁺-dependent fluorescence emission was detected at 535 nm. Dye photobleaching was minimized by restricting the power level of the light source and by preventing exposure to light when data were not being acquired.

Papillary muscles were mounted in a chamber that contained 70 ml of physiologic salt solution maintained at a temperature of 30°C. The muscle was clamped at its lower end to a small plastic block. Electrical stimulation was accomplished with square-wave unipolar pulses of 5 ms duration delivered through platinum field electrodes at 20% above threshold intensity. To reduce the compliance of the preparation, the chorda tendinea was lashed to a thin glass strand (100 μm diameter fiber optic) attached to the arm of a servocontrolled electromagnetic muscle lever operated in the isometric (force) or isotonic (velocity) modes (Model 300B; Cambridge Technology, Inc; Cambridge MA).

Resting muscle length was adjusted to a point where the tension developed during a twitch was optimal.37The diameter and length of the muscle from the clamp to the chorda tendinea were measured in situ . The bathing solution was continuously bubbled with 100% oxygen, buffered with 5 mm 3-[N-morpholino] propanesulfonic acid at pH 7.4, and contained (mM): Na⁺, 140; K⁺, 5; Ca²⁺, 2.0; Mg²⁺, 1; Cl⁻, 103.5; SO^42-, 1; glucose, 10; acetate, 20.

Isometric force was measured at optimal resting length. The rate of change of isometric force (dF/dt) was also calculated by computing the first derivative of isometric force. Unloaded shortening velocity (V^U) was measured by switching the servomotor to isotonic zero-load mode at the onset of the stimulus and recording the shortening rate. Unloaded shortening is reported in units of multiples of the resting muscle length (optimal length).

Skinned trabeculae were prepared by isolating small trabeculae (100–250 μm in diameter, 1.5–2 mm in length) from the right ventricle and rendering the surface membrane permeable to bathing solution constituents by exposing them to a relaxing solution with a pCa (−log[Ca²⁺]) of 9 along with 50% (vol/vol) glycerol plus 1% Triton X-100 (pH 7.0).38Trabeculae were mounted in small aluminum-foil T-shaped clips and attached to two small stainless-steel hooks in a plastic flow-through chamber with a volume of 100 μl. One hook was attached to a micromanipulator and the other to a force transducer (AE801; SensoNor, Horten, Norway). All trabeculae were free of side branches and only those preparations with sharp diffraction patterns were chosen for study. The composition of the solutions was determined according to an iterative computer program that calculated the equilibrium concentration of ligands and ions by use of published affinity constants as outlined previously.39All solutions contained (mM) Imidazole 50, EGTA 10, Mg²⁺1, Mg adenosine triphosphate 5, creatine phosphate 15, pH 7.0, ionic strength 150, along with 20 units/ml creatine phosphokinase.

In all experiments an inline calibrated anesthetic vaporizer was used to add the appropriate concentration of isoflurane or sevoflurane to the preparation. Concentrations of volatile anesthetics in the gas over the bathing solution were monitored by Raman spectroscopy (Rascal II, Ohmeda, Madison, WI) and were also verified in the bathing solution by gas chromatography (5880A, Hewlett-Packard, Palo Alto, CA). The 1 minimum alveolar concentration (MAC) values for isoflurane (Abbott Laboratories; North Chicago, IL) in the ferret at 23°C and 30°C were calculated from the 1 MAC value at 37°C (1.5% gas phase) because the MAC values at different temperatures are essentially constant when expressed as aqueous phase concentrations. The same procedure was used to determine the 1 MAC value for sevoflurane (Abbott Laboratories) at 23°C and 30°C except that the 1 MAC value at 37°C in the rat was used.

Content of contractile proteins was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Samples of tissue were homogenized in a buffer solution containing KCl (100 mm) and Tris-HCl (10 mm, pH 6.0) and aliquots were then diluted with sample buffer solution containing Tris-HCl (200 mm), sodium dodecyl sulfate (1%), EDTA (0.1 mm), glycerol (5%), dithiothreitol (26 mm), pH 8.0 and boiled for 2 min. After heating, additional dithiothreitol was added and samples were loaded onto precast mini-gels (4–15% Ready Gel, Bio-Rad, Hercules, CA) and run at 200V for 1 h at room temperature. They were then rinsed three times for 5 min in double-distilled water and stained with Bio-Safe Coumassie dye (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Porcine cardiac myosin (Sigma-Aldrich, St. Louis, MO) was used as a standard. Band density was normalized to that of the actin band obtained for each sample. The Western blot technique was used to determine the content of SERCA, NCX, and PMCA. The same procedure was followed except gels were not stained with Coumassie dye. Instead, proteins were transferred to polyvinylidene fluoride membrane (Amersham Biosciences, Piscataway, NJ) at 100 V for 1 h at 4°C. The membrane was blocked at 4°C overnight with 5% nonfat dry milk and incubated with the primary antibody (Affinity Bioreagents, Golden, CO) for 1 h at room temperature. The secondary antibody (Amersham Biosciences, Piscataway, NJ) with horseradish peroxidase was added and incubated with the membrane for 1 h at RT. The membrane was then incubated for 5 min in chemiluminescent substrate and the membrane was exposed to radiographic film (Eastman Kodak, Rochester, NY) to detect the signal. The density of the bands was analyzed with NIH Image software (National Institutes of Health, Bethesda, MD). The linearity of response was confirmed by analyzing samples of serial dilutions of the membrane proteins. Bands were normalized by dividing the densitometric units by the amount of protein in each preparation.

Results are reported as the mean ± SD. Force was normalized for muscle cross-sectional area. The length and diameter were measured at peak light intensity, and the muscle was assumed to approximate a cylinder. Statistical significance (P < 0.05) was determined by unpaired Student t  test or one-way or two-way repeated-measures analysis of variance where appropriate (SigmaStat 2.0 for Windows; SPSS Inc., Chicago IL). Post hoc  analysis was performed with Tukey test (P < 0.05).

Table 1summarizes the characteristics of the animal model. There were no apparent differences in mean body weight, left ventricular weight, papillary muscle cross-sectional area, or papillary muscle length between groups. However, as expected, the banded animals had larger right ventricles and the cross-sectional areas of individual cells were increased significantly.

Pulmonary artery banding also produced significant changes in contractile function as shown in figure 1and summarized in table 2. Resting force at optimal length was higher in muscles from banded animals and developed F, dF/dt, and V^Uwere all reduced by approximately 50%. In contrast, the time-to-peak force and the time-to-50% relaxation were not changed by pulmonary artery banding.

Figure 1shows typical records of the calcium transients and force development in papillary muscles from normal and banded animals. Mean resting [Ca²⁺]ⁱwas increased in papillary muscles from banded animals but peak [Ca²⁺]ⁱwas not significantly different between groups (table 2). To assess the function of the sarcoplasmic reticulum to release and resequester Ca²⁺, we measured the timing of the peak of the Ca²⁺transient (primarily release) and its rate of decline (primarily reuptake). In hypertrophied muscles, the peak of the Ca²⁺transient occurred later and its rate of decline was prolonged compared with control muscles (table 2).

To clarify the mechanisms responsible for these alterations in the Ca²⁺transient, we performed sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblot analysis to investigate the level of expression of the three main pumps involved in Ca²⁺homeostasis in the heart (SERCA, NCX, and PMCA). Figure 2shows the results of Western immunoblot analysis of the level of expression of the primary isoforms of these proteins expressed in the heart (PMCA2a, NCX1, and SERCA2a). We found that both PMCA2a and SERCA2a were moderately decreased in muscles from banded animals (n = 8, P < 0.05) and note that this difference is consistent with the moderate prolongation of the Ca²⁺transient seen in figure 1A.

Figure 3Asummarizes the response of peak isometric force to isoflurane and sevoflurane at 0.5 MAC and 1 MAC. Isoflurane and sevoflurane produced a similar dose-dependent decrease in peak isometric force in both types of muscle. Both anesthetics also produced a dose-dependent decrease in time to peak force and time to 50% relaxation (data not shown). In a similar manner, these anesthetics decreased dF/dt and V^Udose-dependently in both types of muscle although the effects on shortening were of lesser magnitude (figs. 3B and 3C).

Figure 4summarizes the effects of 1 MAC anesthetic on the three indices of contractility and peak systolic [Ca²⁺] in normal and hypertrophied heart. Both isoflurane and sevoflurane produced a greater decrease in developed force in hypertrophied muscles, but there was not a significant difference in this effect between anesthetics (isoflurane: normal =−36.0 ± 9.9%, hypertrophy =−48.8 ± 8.5%; sevoflurane: normal =−29.5 ± 8.3%, hypertrophy =−47.2 ± 10.9%). Similarly, both agents also produced a greater decrease in dF/dt in hypertrophied muscle, but the difference between anesthetics in normal muscle did reach statistical significance (isoflurane: normal =−31.6 ± 9.0%, hypertrophy = 41.8 ± 7.5%; sevoflurane: normal =−23.5 ± 7.5%, hypertrophy =−37.8 ± 7.9%). In addition, V^Udecreased to a greater extent in hypertrophied muscles in the presence of either anesthetic but there was no difference between anesthetics in the same type of muscle (isoflurane: normal =−18.3 ± 9.1%, hypertrophy =−33.1 ± 11.2%; sevoflurane: normal =−14.7 ± 9.4%, hypertrophy =−28.7 ± 10.4%).

The severe reduction in baseline force in the muscles from banded animals (fig. 1and table 2) lead us to evaluate the level of expression of several of the proteins that are important for normal contraction. These results are summarized in figure 5and establish that there were no significant differences in the expression of actin, myosin heavy chain, tropomyosin, troponin C, troponin I, troponin T, and myosin light chains 1 and 2.

The effects of both isoflurane and sevoflurane on [Ca²⁺]ⁱas determined by aequorin in isolated papillary muscles are summarized in figure 6. Figure 6Ashows the response of resting [Ca²⁺]ⁱto increasing doses of isoflurane and sevoflurane. In general, there were no obvious changes in resting [Ca²⁺] in the presence of either anesthetic in either type of muscle. However, in contrast to normal muscles, the mean resting [Ca²⁺]ⁱin hypertrophied muscles decreased over time and was observed to decrease to less than the control value upon washout of both anesthetics (P < 0.05).

Both agents decreased peak [Ca²⁺]ⁱ, and there was no obvious difference between them (fig. 6B). The effect of both anesthetics on [Ca²⁺]ⁱonly reached statistical significance at 1 MAC in normal muscles. In papillary muscles from banded animals, isoflurane produced a decrease in [Ca²⁺]ⁱat both concentrations, whereas the response to sevoflurane only reached statistical significance at a dose of 1 MAC. In contrast to the effects on contractility, neither agent appeared to cause a greater decrease in peak [Ca²⁺]ⁱin muscles from hypertrophied hearts.

Because of the central role of peak [Ca²⁺]ⁱin excitation-contraction coupling in the heart and because the aequorin signal is inherently somewhat noisy and requires signal averaging (fig. 1), we also carried out experiments using a different Ca²⁺indicator (fluo-3) in isolated myocytes. For these experiments we focused on the effects of isoflurane and sevoflurane at 1 MAC (fig. 6C). Using this technique, both agents did produce a significantly greater depression of [Ca²⁺]ⁱin cells from banded animals, indicating that decreased [Ca²⁺]ⁱplays a role in the differential effect in hypertrophy.

Figure 7summarizes the results of experiments to evaluate the baseline Ca²⁺sensitivity in normal and hypertrophied heart and the response to 1 MAC isoflurane. We found that baseline Ca²⁺sensitivity was significantly lower (pCa⁵⁰: control = 5.61 ± 0.05; banded = 5.09 ± 0.03) and the steepness of the relationship was greater (Hill coefficient: normal = 2.34 ± 0.29; banded = 3.81 ± 0.51) in trabeculae from banded animals. In the presence of 1 MAC isoflurane the calcium sensitivity of both groups decreased (pCa⁵⁰iso: normal = 5.42 ± 0.06; banded = 4.99 ± 0.03) but the slope remained unchanged. The dashed lines in figure 7illustrate the effect that a simultaneous decrease in [Ca²⁺] and Ca²⁺sensitivity could have on force production. In this example, an equal change in [Ca²⁺] can produce a disproportionate change in relative force in hypertrophied heart muscle. Of course, a greater decrease in [Ca²⁺]ⁱwould only be expected to exaggerate the disparity.

Although myocardial hypertrophy is generally considered to be a compensatory process, its progression can lead to varying degrees of decreased contractility and at the extreme can result in heart failure. Intraoperatively, any additional reduction in contractility resulting from the anesthetic is likely to adversely affect systemic hemodynamics, as both myocardial and autonomic reflex responses to hypotension are blunted in patients with hypertrophy and heart failure.24,25,40,41This study illustrates that isoflurane and sevoflurane both produce an exaggerated decrease in the contractility of isolated hypertrophied myocardium. In addition, this study characterizes the effects of volatile anesthetic agents on the intracellular Ca²⁺transient in hypertrophy and suggests that baseline Ca²⁺sensitivity and the slope of the relationship between [Ca²⁺] and force may be altered in such a way that exaggerates the depression caused by decreased Ca²⁺availability.

All commonly used volatile anesthetics including halothane, enflurane, isoflurane, and the newer agents sevoflurane and desflurane depress the contractile force of the heart. Studies in isolated heart muscle preparations have consistently demonstrated that these agents produce concentration-dependent reversible decreases in contractility. Of the anesthetics mentioned above, halothane causes the greatest depression, enflurane the second greatest depression, and isoflurane the least depression. The effects of desflurane and sevoflurane are reportedly similar to those of isoflurane.42–44The mechanisms involved in this negative inotropic effect are incompletely understood, but the bulk of experimental evidence suggests that the volatile anesthetics exert a negative inotropic effect predominantly by reducing the amount of intracellular Ca²⁺released each beat. What is still relatively unclear is to what degree the many processes involved in Ca²⁺homeostasis are inhibited and to what extent the alteration of these mechanisms by disease influences the overall effects of these drugs.

In this study we used a model of right ventricular pressure-overload hypertrophy in ferrets. Tissue from the right ventricle is commonly studied because the right ventricle generally possesses relatively long and thin papillary muscles that are ideal for mechanical studies of isolated heart muscle. In our model, banding of the pulmonary artery produced significant hypertrophy, as documented by increased weight of the right ventricle relative to the left and increased cell size. Baseline contractility was lower in papillary muscles from banded animals; this is also consistent with previous reports.29–32This was documented by the reduction in peak isometric force, peak dF/dt, and V^U. On the other hand, the time course of the twitch (time to peak force and time to 50% force) was unchanged by hypertrophy.

We examined the effects of 0.5 MAC and 1.0 MAC isoflurane and sevoflurane on three measures of contractility and found that both volatile anesthetic agents decrease contractility in a dose-dependent manner in normal and hypertrophied heart. In addition, both agents appear to produce approximately equal depression. On the other hand, we found that isoflurane and sevoflurane depress contractility to a greater extent in pressure-overloaded heart muscle. In the case of isoflurane, this is consistent with past reports for isolated heart muscle preparations.21However, although this phenomenon was documented many years ago, its underlying basis is unknown.

One potential mechanism by which isoflurane and sevoflurane might depress myocardial contractility to a greater extent in pressure-overload hypertrophy is via  relatively selective inhibition of just a subset of all the processes involved in Ca²⁺homeostasis. As pointed out in the introduction, halothane appears to inhibit Ca²⁺influx across the surface membrane and also releases Ca²⁺from the sarcoplasmic reticulum, depleting the cell of calcium. Isoflurane, on the other hand, primarily limits Ca²⁺influx and maintains sarcoplasmic reticulum Ca²⁺content.27,28This difference probably explains the greater myocardial depression of contractility in normal heart caused by an equianesthetic concentration of halothane.

In addition, we speculate that this dissimilarity also helps to explain the increased susceptibility of hypertrophied heart to anesthetics like isoflurane. To explain this effect, it is necessary to note that in hypertrophy and heart failure the function of the sarcoplasmic reticulum is reduced,31,45–47whereas the surface membrane Ca²⁺channels seem to be relatively spared.48,49The changes we observed in the Ca²⁺transient are consistent with this process and suggest that in hypertrophy and heart failure, ventricular myocytes are more dependent on Ca²⁺influx for activation. In this case, the exaggerated depression by volatile anesthetics like isoflurane may result from inhibiting a mechanism (Ca²⁺influx) that has become more important in the presence of a pathologic process like myocardial hypertrophy.

Significant changes in the function of the sarcoplasmic reticulum are reflected in characteristic changes in the intracellular Ca²⁺transient shown in figure 1and in reports by other investigators.1,45,50We also found that the resting [Ca²⁺]ⁱwas higher in hypertrophied heart, as reported by others.51In addition, pressure overload caused the time course of the intracellular Ca²⁺transient to slow such that peak [Ca²⁺]ⁱwas reached later and the rate of decline was reduced, as has also been documented previously.31,32The decrease in SERCA2a levels by the Western immunoblot technique further supports this conclusion,.47,52,53In addition, we show that the level of expression of PMCA2a is decreased in hypertrophied hearts. Although the function of PMCA in the heart has been questioned,54it is plausible that this change could contribute to the increased resting [Ca²⁺]ⁱ. Another mechanism that might contribute to the prolongation of the calcium transient is the decreased Ca²⁺sensitivity observed in muscles from banded animals (fig. 7). One might expect less Ca²⁺to bind to troponin C in this setting, and the free [Ca²⁺]ⁱwould therefore be expected to increase. It seems less likely that this is the predominant mechanism, however, as the peak of the Ca²⁺transient would also be expected to increase.

In the presence of both anesthetics at 1 MAC, we found that peak intracellular [Ca²⁺] decreased significantly in normal and hypertrophied heart. On the other hand, at 0.5 MAC sevoflurane, the peak [Ca²⁺]ⁱin muscles from banded animals seemed to increase when compared with the response observed in hypertrophied muscles in the presence of 0.5 MAC isoflurane. This response appears to be restricted to peak [Ca²⁺] in the hypertrophied muscles in the presence of sevoflurane and suggests that sevoflurane may somehow potentiate sarcoplasmic reticulum Ca²⁺release at low concentrations. It is noteworthy that sevoflurane has been reported to increase sarcoplasmic reticulum Ca²⁺content in isolated myocytes by inhibiting the PMCA,28but it is difficult to tie this mechanism directly to the increase in Ca²⁺release observed here at 0.5 MAC.

However, this study does suggest that a decrease in Ca²⁺availability is at least partially responsible for the increased susceptibility of hypertrophied myocardium to anesthetic-induced depression at 1 MAC. We encountered some difficulty in establishing this relationship because it was not reflected in a statistically greater decrease in peak intracellular [Ca²⁺] as measured by aequorin. One plausible explanation for this is that the noisiness of the aequorin technique may have obscured the difference. This complication results from the fact that the aequorin light signal is generally very weak and necessitates the use of high sensitivity photomultipliers. Aequorin is, however, still a useful Ca²⁺indicator for work in isolated muscle because it is not prone to motion artifacts and tends to remain confined to the cytoplasm.36It is unlikely that the sensitivity of aequorin has anything to do with this because the peak signals obtained during a twitch are well within the linear region of the aequorin calibration curve.36However, we were able to show that the relative decrease in [Ca²⁺]ⁱdid reach statistical significance in isolated myocytes from banded animals when monitored by fluo-3, and this data compliments the data obtained in papillary muscles with aequorin.

Although we believe that this difference alone is of physiologic relevance, our data also suggests that volatile anesthetic effects on intracellular [Ca²⁺] are probably not the only mechanism involved in the increased susceptibility to depression in pressure-overload hypertrophy. Ca²⁺sensitivity has been reported to decrease in hypertrophy and heart failure,55–57and isoflurane has also been reported to decrease it in normal heart primarily at concentrations of 1 MAC and above.8,58In this study we found that Ca²⁺sensitivity was altered to a greater extent by the disease process itself and interpret our data as suggesting that altered Ca²⁺sensitivity plays a significant role both in the baseline depression of contractility and the exaggerated response to volatile anesthetics.

One way to explain how Ca²⁺sensitivity changes can be responsible for increased myocardial depression in hypertrophy is to recognize that hypertrophied muscles may operate on a steeper pCa-force curve that is shifted to the right. If this is true, as suggested by this study, then an equal change in intracellular [Ca²⁺] could decrease contractility to a greater extent (fig. 7). This explanation does not require a greater effect of anesthetic on the pCa-force relationship. For example, if the [Ca²⁺]ⁱwere to decrease from pCa 5.2 to 5.4 (0.2 pCa units) in both types of muscle then according to the pCa-force relationship the isometric force in normal muscles would be expected to decrease from 0.83 to 0.55. This would yield a percentage decrease of 33% (dashed lines in fig. 7, panel A). In comparison, the same change in [Ca²⁺]ⁱin hypertrophied muscles would be expected to decrease isometric force from 0.30 to 0.08 for a percentage decrease of 73% (dashed lines in fig. 7, panel B). In this example, most of the difference in force is set up by the baseline shift of the curve to the right and the increased slope rather than the change in sensitivity caused by the anesthetic.

It is not possible to make a direct correlation between the measurements obtained in skinned fibers and intact papillary muscles. This is a result of the difficulty in comparing [Ca²⁺]ⁱmeasured in intact muscle preparations with the response of skinned preparations because the skinning process or the solutions used in these measurements seem to give different readings of Ca²⁺sensitivity.37,59However, taken together our data suggest that changes in [Ca²⁺]ⁱand altered calcium sensitivity are both responsible for the exaggerated effects of isoflurane and sevoflurane on contractility in pressure-overload hypertrophy. This data also suggests that any intervention that produces ionized hypocalcemia, such as the administration of citrated blood products, might be expected to have particularly deleterious effects on contractility in the setting of underlying myocardial hypertrophy and heart failure.