Propofol and Ketamine Only Inhibit Intracellular Ca²⁺Transients and Contraction in Rat Ventricular Myocytes at Supraclinical Concentrations 

Isolated adult ventricular myocytes from rat hearts were obtained as previously described. [17]The hearts were excised, cannulated via the aorta, attached to a modified Langendorff perfusion apparatus, and perfused with oxygenated (95% O², 5% CO²) Krebs-Henseleit buffer (37 [degree sign] Celsius) containing the following (in mM): 118 NaCl, 4.8 KCl, 1.2 MgCl², 1.2 KH²PO sub 4, 1.2 CaCl², 37.5 NaHCO³, and 16.5 dextrose (pH 7.35). After a 5-min equilibration period, the perfusion buffer was changed to a Ca²+-free Krebs-Henseleit buffer containing 30 mg of collagenase type II (Worthington Biochemical Corp., Freehold, NJ; lot no. M6C152; 347 U/ml). After digestion of collagenase (20 min), the ventricles were minced and shaken in Krebs-Henseleit buffer. The resulting cellular digest was washed, filtered through cheese cloth, and resuspended in phosphate-free HEPES-buffered saline containing the following (in mM): 118.0 NaCl, 4.8 KCl, 1.2 MgCl², 1.25 CaCl², 11.0 dextrose, 25.0 HEPES, and 5.0 pyruvate (pH 7.35). This combination was bubbled vigorously immediately before use with 100% O². Typically, 6–8 x 10 sup 8 cells per rat heart were obtained using this procedure. Viability, as assessed by the percentage of cells retaining a rod shape with no blebs or granulations, was routinely 80–90%. Myocytes were suspended in HEPES-buffered saline (1 x 10⁶cells/ml) and stored in an oxygen hood until used.

For simultaneous measurement of contraction and [Ca²+] sub i, ventricular myocytes (0.5 x 10⁸cells/ml) were incubated in HEPES-buffered saline containing 2 micro Meter fura-2/acetoxymethyl ester at 37 [degree sign] Celsius for 20 min. Fura-2-loaded ventricular myocytes were placed in a temperature-regulated (28 [degree sign] Celsius) chamber (Bioptechs, Inc., Butler, PA) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope (Olympus America, Lake Success, NY). We used a chamber temperature of 28 [degree sign] Celsius because it allowed us to achieve stable reproducible contractile responses without any spontaneous contractions or Ca²+ overload of the cell throughout the entire experimental protocol. The volume of the chamber was 1.5 ml. The cells were superfused continuously with HEPES-buffered saline at a flow rate of 2 ml/min and field-stimulated via bipolar platinum electrodes at a frequency of 0.3 Hz (60 volts) for 5 ms using a Grass SD9 stimulator (Grass Instruments, West Warwick, RI). Myocytes [approximately] 110 micro meter in length were chosen for study according to the following criteria:(1) rod-shaped appearance with clear striations and no membrane blebs;(2) a negative staircase of twitch performance on stimulation from rest; and 3) the absence of spontaneous contractions.

Fluorescence measurements were performed on single ventricular myocytes using a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, South Brunswick, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The cells were also illuminated with red light at a wavelength > 600 nm for simultaneous video edge detection. An additional postspecimen dichroic mirror deflected light at wavelengths > 600 nm into a charge coupled device video camera (Phillips VC 62505T; Marshall Electronics, Culver City, CA) for measurement of myocyte shortening and relengthening. The fluorescence sampling frequency was 100 Hz, and data were collected using the Felix software package from Photon Technology International (South Brunswick, NJ). Because of technical difficulties in calibrating fura-2 in single cardiac myocytes, [Ca²+]ⁱwas estimated by comparing the cellular fluorescence ratio with fluorescence ratios acquired using fura-2 (free acid) in buffers containing known Ca²+ concentrations. [18] 

Simultaneous measurement of cell shortening was monitored using a video edge detector (Crescent Electronics, Sandy, UT) with a 16-ms temporal resolution. The video edge detector was calibrated using a stage micrometer so that cell lengths during shortening and relengthening could be monitored. Myocytes typically contracted on one end with the other end lightly attached to the chamber. Thus, contraction represented unloaded isotonic shortening. LabVIEW[registered sign](National Instruments, Austin, TX) was used for data acquisition of cell shortening using a sampling rate of 100 Hz.

Fluorescence data for the measurement of [Ca²+]ⁱwere imported into LabVIEW[registered sign], where the [Ca²+]ⁱand myocyte contractile responses were analyzed synchronously and simultaneously. The following parameters were calculated for each individual contraction: diastolic [Ca²+]ⁱand cell length; systolic [Ca²+]ⁱand cell length; changes in [Ca²+]ⁱand twitch amplitude; time to peak [Ca²+]ⁱand peak shortening; time to 50% diastolic [Ca²+]ⁱand 50% relengthening. Parameters from 15 contractions were averaged to obtain mean values at baseline and in response to the various interventions. Averaging the parameters over time minimizes beat-to-beat variation.

Myocyte length in response to field stimulation was measured in microns and is expressed as the change from resting cell length (twitch amplitude). Changes in twitch amplitude in response to the interventions are expressed as a percent of baseline shortening. Changes in timing were measured in milliseconds and normalized to changes in amplitude. Changes in [Ca²+]ⁱwere measured as the change in the 340/380 ratio from baseline. Changes in the 340/380 ratio in response to an intervention are expressed as a percent of the control value in the absence of any intervention.

Protocols were designed such that each cell could be used as its own control.

Protocol 1. Changes in myocyte shortening and [Ca²+]ⁱduring exposure to propofol, Intralipid[registered sign](Kabi Pharmacia, Clayton, NC), or ketamine were determined. Baseline measurements were collected from individual myocytes for 1.5 min in the absence of any intervention. Myocytes were exposed to four concentrations of each anesthetic agent (30, 100, 300, and 1,000 micro Meter) by exchanging the buffer in the dish with new buffer containing the intravenous anesthetic agent at the desired concentration. Propofol was applied in its commercially available 10% Intralipid[registered sign] emulsion (56 mM [10 mg/ml] propofol, 10% soybean oil, 2.25% glycerol, 1.2% purified phospholipid). Data were acquired for 1.5 min after a 5-min equilibration period in the presence of the anesthetic agent. The effect of the solvent of propofol, Intralipid[registered sign], was examined at concentrations equivalent to those used for the propofol-Intralipid mixture. Individual myocytes were exposed only to one intravenous anesthetic agent or the Intralipid[registered sign] vehicle.

Protocol 2. To determine whether propofol or ketamine alters release of Ca²+ from intracellular Ca²+ stores, we measured caffeine-induced release of Ca²+ in the presence or absence of the intravenous anesthetic agent. Baseline [Ca²+]ⁱwas measured in individual myocytes for 1.5 min. Propofol (100 or 1,000 micro Meter), ketamine (100 or 1,000 micro Meter), nifedipine (1 micro Meter, which completely blocks L-type Ca²+ currents), or ryanodine (10 micro Meter, which activates the Ca²+ release channel and depletes intracellular Ca²+ stores) were then added to the superfusion buffer and allowed to equilibrate for 5 min. Field stimulation of the myocyte was discontinued, and caffeine (20 mM) was applied to the cell 15 s later. The amplitude of the [Ca²+]ⁱtransient induced by caffeine was compared with the amplitude of the field-stimulated [Ca²+]ⁱtransient before addition of the respective drugs and is reported as a percent of the control amplitude.

Protocol 3. To determine whether propofol or ketamine alters myofibrillar Ca²+ sensitivity, we examined the dose-response curve to increasing concentrations of extracellular Ca²+([Ca²+]^o) in the presence or absence of an intravenous anesthetic agent. Baseline parameters were collected from individual myocytes for 1.5 min. Dose-response curves for [Ca²+]^owere performed by exchanging the buffer in the dish with a new buffer with the desired [Ca²+] sub o. Data were acquired for 1.5 min after establishment of a new steady state. Dose-response curves for [Ca²+]^owere then performed in the presence of either nifedipine (10 nM), propofol (100 micro Meter), or ketamine (100 micro Meter). Cells were allowed to stabilize for 5 min after addition of each agent.

We have verified that propofol and ketamine have no effect on the fura-2 signal at the concentrations tested. This was confirmed using fura-2 free acid in HEPES buffer and by examining whether either agent altered the fluorescence ratio.

Propofol, Intralipid[registered sign], and ketamine were obtained from the Cleveland Clinic Pharmacy (Cleveland, OH). Caffeine was purchased from Sigma Chemical Co. (St. Louis, MO). Nifedipine and ryanodine were obtained from Research Biochemicals, Inc. (Natick, MA).

Each experimental protocol was performed on multiple myocytes from the same heart and repeated in at least four hearts. Results obtained from myocytes in each heart were averaged so that all hearts were weighted equally. The dose-dependent effects of propofol, Intralipid[registered sign], or ketamine on myocyte shortening and [Ca sup 2+]ⁱwere assessed using one-way analysis of variance with repeated measures and the Bonferroni/Dunn post hoc test. Comparisons between groups were made by two-way analysis of variance. Results are expressed as the mean +/- SEM. Differences were considered statistically significant at P < 0.05.

Baseline parameters were stable over the time course of these experiments. Baseline [Ca²+]ⁱwas 80 +/- 12 nM, and the diastolic cell length was 112 +/- 2 micro meter. Peak [Ca²+]ⁱwas 350 +/- 23 nM. Twitch amplitude was 10%(11.2 +/- 0.5 micro meter) of the baseline diastolic resting cell length. Time to peak concentration (Tp) of [Ca²+]ⁱand shortening were 147 +/- 3 and 184 +/- 3 ms, respectively. Time to 50% recovery (Tr) for [Ca²+]ⁱand shortening were 322 +/- 5 and 311 +/- 3 ms, respectively.

(Figure 1(A)) demonstrates that addition of propofol to a single, field-stimulated ventricular myocyte results in dose-dependent inhibition of myocyte shortening and a concomitant decrease in peak [Ca sup 2+]ⁱ. Individual contractions and [Ca²+]ⁱtransients are illustrated in Figure 1(B). Propofol had no effect on resting [Ca sup 2+]ⁱor cell length. The summarized data are shown in Figure 2. Propofol caused dose-dependent decreases in myocyte shortening and peak [Ca²+]ⁱ. The Intralipid[registered sign] vehicle had only small inhibitory effects on myocyte shortening and peak [Ca²+]ⁱat concentrations equivalent to those obtained with 300 micro Meter propofol.

Propofol prolonged both Tp and Tr for shortening and [Ca²+]ⁱat concentrations > 100 micro Meter (Figure 3). The Intralipid[registered sign] vehicle had no effect on Tp or Tr at any concentration tested.

(Figure 4(A)) demonstrates that addition of ketamine to a single, field-stimulated ventricular myocyte results in dose-dependent inhibition of myocyte shortening and a concomitant decrease in peak [Ca sup 2+]ⁱ. Individual contractions and [Ca²+]ⁱtransients are shown in Figure 4(B). Ketamine had no effect on resting [Ca²+]ⁱor cell length. The summarized data are shown in Figure 5. At concentrations > 100 micro Meter, ketamine caused dose-dependent decreases in shortening and peak [Ca²+]ⁱ.

Ketamine had no significant effect on Tp or Tr for shortening or [Ca²+]ⁱat concentrations up to 300 micro Meter (Figure 6). Ketamine prolonged both Tp and Tr for myocyte shortening at the highest concentration tested. Tp and Tr for [Ca²+]ⁱwere increased at this concentration, but these changes were not statistically significant (P = 0.09 for both Tp and Tr).

We assessed the extent to which propofol or ketamine altered the amount of Ca²+ released from the SR in response to caffeine (20 mM). Neither propofol (100–1,000 micro Meter), ketamine (100–1,000 micro Meter), nor nifedipine (1 micro Meter) altered the amplitude of the caffeine-releasable pool of Ca²+ compared with that observed with the control response to caffeine (Figure 7). In contrast, ryanodine (10 micro Meter), which activates the Ca²+ release channel and depletes intracellular stores, abolished the caffeine-induced increase in [Ca²+]ⁱ.

The effects of [Ca²+]^oon myocyte shortening and [Ca²+]ⁱwere examined in the presence and absence of intravenous anesthetic agents. Increasing [Ca²+]^ofrom 1 to 4 mM (without any intravenous anesthetic agents) resulted in a dose-dependent increase in shortening and a concomitant increase in peak [Ca²+]ⁱ(Figure 8). The L-type voltage-gated Ca²+ channel blocker nifedipine (10 mM) had no significant effect on the dose-response curve for [Ca²+]^ocompared with control; however, propofol (100 micro Meter) but not ketamine (100 micro Meter) caused a leftward shift in the dose-response curve for [Ca²+]^ofor shortening with no concomitant effect on peak [Ca²+]ⁱ.

Increasing [Ca²+]^owithout an intravenous anesthetic agent (control) resulted in dose-dependent decreases in Tp and Tr for both myocyte shortening and [Ca²+]ⁱ(Figure 9). Neither propofol (100 micro Meter) nor ketamine (100 micro Meter) had a significant effect on the changes in Tp or Tr for myocyte shortening and [Ca²+]ⁱin response to increasing [Ca²+]^o(Figure 9).

The current study is the first to assess simultaneously the effects of intravenous anesthetic agents on myocyte shortening and [Ca sup 2+]ⁱat the cellular level. Although the clinical effects of propofol on the cardiovascular system have been reported to result largely from reductions in systemic vascular resistance and diminished preload, [3,4]our results demonstrate that propofol can exert a direct negative inotropic effect on isolated rat ventricular myocytes and that this is at least partly mediated by a reduction in peak [Ca²+]ⁱ.

The clinical relevance of this negative inotropic effect of propofol is somewhat doubtful. The peak plasma concentration of propofol after bolus administration has been estimated at 50 micro Meter. [19]In normal circumstances, the steady-state free plasma concentration of propofol likely would not exceed 1–2 micro Meter because 97–98% binds to serum protein. [20]Calculations of plasma concentration in vivo, however, are difficult, because the rate of exchange among propofol-containing liposomes, the aqueous phase, serum proteins, and other cellular constituents is not precisely known. In addition, protein binding of propofol is unlikely to be instantaneous in vivo, so the free drug concentration associated with a bolus injection would probably be higher than the steady-state value. In this study, propofol was administered in its commercially available 10% Intralipid[registered sign] emulsion to achieve chamber concentrations of 30, 100, 300, and 1,000 micro Meter. Propofol (100 micro Meter) caused a 50% decrease in myocyte shortening and a concomitant 35% decrease in the amplitude of the intracellular Ca²+ transient. This result is consistent with the negative inotropic effect observed with the same dose of propofol in guinea pig [12]and ferret [8]ventricular myocardium. Propofol (100 micro Meter) caused only a 24% decrease, however, in left ventricular systolic pressure in isolated rabbit hearts [9]and had little or no effect on isometrically contracting rat papillary, muscles. [11]Because we saw effects only at high concentrations, it is unlikely that propofol or ketamine in clinically relevant concentrations exert their effects via this mechanism. Given the uncertainty in calculating the in vivo concentration of propofol, however, and the likelihood that the concentration would be higher when serum protein concentration is reduced (hemodilution, liver disease), it is difficult to determine the upper limit for clinically relevant concentrations of propofol. Moreover, sensitivity to the negative inotropic effect of propofol has been shown recently to be increased in the setting of congestive heart failure. [21] 

We also examined the effects of the Intralipid[registered sign] vehicle on myocyte shortening and [Ca²+]ⁱ. Intralipid[registered sign] is primarily a fatty acid that combines with glycerol and lecithin to create micelles for transporting propofol. Because unsaturated fatty acids can enhance contractile properties of cardiac myocytes, [22]it was conceivable that the Intralipid[registered sign] vehicle made for a more potent negative inotropic effect of propofol. The effects of the Intralipid[registered sign] vehicle on shortening and [Ca²+]ⁱwere minimal, however, in the wrong direction (negative), and apparent only at concentrations equivalent to those obtained with >or= to 300 micro Meter propofol.

The inotropic effects of ketamine on myocardial performance are controversial. Ketamine has been shown to exert either an indirect positive inotropic effect or a direct negative inotropic effect. [13,14,23,24]. At clinically relevant concentrations, ketamine has been shown to exert a positive inotropic effect via inhibition of neuronal catecholamine uptake and subsequent beta-adreno-receptor activation. [13,23]In contrast, ketamine has been shown to depress contractility directly in isolated cardiac tissue from rabbit [25]and ferret. [14]Ketamine also has been shown to cause both positive and negative inotropic effects in isolated rat papillary [5]and atrial muscle [26]depending on the concentration used. In the current study, the 50% depression of shortening by ketamine (200 micro Meter) is similar to a previous report of 50% depression of left ventricular pressure in isolated guinea pig hearts [12]or isometric tension in ferret ventricular strips. [4]In the clinical setting, however, the total concentration of ketamine in plasma after intravenous bolus injection is 60 micro Meter, [2]resulting in a steady-state free plasma concentration of 50 micro Meter because of 12% protein binding. [27]Thus, the direct inhibitory effects of ketamine on myocyte shortening and [Ca²+]ⁱthat we observed were apparent only at concentrations higher than are normally encountered in the clinical setting.*

We also examined whether propofol or ketamine caused changes in the timing of myocyte shortening or [Ca²+]ⁱconcentration. Changes in the timing parameters could suggest alterations in Ca²+ release in the SR, reuptake, or myofilament Ca²+ sensitivity. Inhibition of the SR Ca²+ pump with thapsigargin has been shown to diminish peak [Ca²+]ⁱconcentration and to prolong Tp and Tr for the [Ca²+]ⁱtransient. [28]Our results demonstrate that propofol but not Intralipid[registered sign] increased Tp and Tr for both myocyte shortening and [Ca²+]ⁱ. In contrast, ketamine had little or no effect on these timing parameters. A propofol-induced inhibition in the rate of Ca²+ uptake in the SR (but no effect on net uptake) is a potential mechanism to explain the prolongation of the timing parameters. Alternatively, an increase in myofilament Ca²+ sensitivity, as our results suggest (Figure 8), could explain the increase in Tr, i.e., Ca²+ could remain bound to the myofilaments longer (decreased off-rate of Ca²+ from troponin C), thereby prolonging the increase in [Ca²+]ⁱconcentration and the contraction. In these conditions, no effect on net Ca²+ uptake by the SR or net Ca²+ content in the SR would have to occur. Another explanation is that the prolongation of Tr is secondary to the measured reduction in the peak [Ca²+]ⁱtransient, [29]because it has been demonstrated that the rate of the [Ca²+]ⁱtransient is dependent on the amount of Ca²+ released. Because Ca²+ uptake by the Ca²+ adenosinetriphosphatase in the SR occurs according to Michaelis-Menton binding, a smaller peak [Ca²+]ⁱcould result in a longer Tr. Further studies using isolated SR vesicles are required to confirm these hypotheses.

Another possible explanation for a decrease in peak [Ca²+]ⁱis a decrease in the amount of Ca²+ released from the SR. Caffeine, which is known to trigger release of Ca²+ from cardiac SR via an interaction with the ryanodine receptor, was used to investigate potential interactions of intravenous anesthetic agents on SR Ca²+ release channels. Application of caffeine (20 mM) to quiescent myocytes resulted in a peak [Ca²+]ⁱsimilar in magnitude to that observed with field stimulation. Peak [Ca²+]ⁱwas not altered by pretreatment with nifedipine, an L-type Ca²+ channel antagonist, but was completely abolished by ryanodine (10 micro Meter), which activates the Ca²+ release channel and depletes intracellular Ca sup 2+ stores at this concentration. Neither propofol nor ketamine altered peak [Ca²+]ⁱeven at the highest concentrations used in this study. These data are consistent with the previously reported inability of ketamine to alter the function of cardiac SR Ca²+ release channels. [30]Our results indicate that neither propofol nor ketamine exerts their negative inotropic effects by altering Ca²+ content in the SR.

Alterations in the sensitivity of the myofilaments to Ca²+ could also modulate the cardiac inotropic response to intravenous anesthetic agents. To examine whether alterations in myofilament Ca²+ sensitivity played a role in the actions of intravenous anesthetic agents, dose-response curves to [Ca²+]^owere generated. Propofol but not ketamine caused a leftward shift in the dose-response curve for [Ca²+]^ofor shortening, with no concomitant effect on peak [Ca²+]ⁱ. We postulated that this was due to an inhibitory effect of propofol on L-type Ca²+ channels, [31]which was overcome by increasing [Ca²+]^o. Nifedipine, however, unlike propofol, failed to alter the dose-response curve to [Ca²+] sub o compared with untreated controls. These results indicate that propofol may increase myofilament Ca²+ sensitivity, which may partially offset the propofol-induced reduction in peak [Ca²+]ⁱ.

One potential limitation of the study is the use of rat cardiomyocytes as a model for human cardiomyocytes, as species differences may exist. In addition, the validity of interpreting externally unloaded myocyte shortening as an indicator of the inotropic state of the myocardium has been reviewed recently. [32]In the intact heart, myocytes normally are exposed to a time-varying external load. In contrast, isolated myocytes are not externally loaded, and the force developed during contraction is unknown. Even without an external load, however, isolated myocytes are shortening against an internal load composed of several components. [33]Despite this limitation, the use of isolated myocytes as a model of cardiac function provides important insight into mechanisms of regulation of excitation-contraction coupling. It should be noted that there has been remarkable consistency among results obtained from unloaded cells and multicellular preparations.

The authors thank Beth Summers and Cindy Shumaker for their excellent technical support and Ronnie Sanders for outstanding work in preparing the manuscript.