Propofol Attenuates β-Adrenoreceptor–mediated Signal Transduction via  a Protein Kinase C–dependent Pathway in Cardiomyocytes

All experimental procedures and protocols were approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Cleveland, OH).

Ventricular myocytes were freshly isolated from adult male Sprague-Dawley rat hearts as previously described. 7Immediately after euthanasia, the hearts were rapidly removed and perfused in a retrograde manner at a constant flow rate (8 ml/min) with oxygenated (95% O²–5% CO²) Krebs-Henseleit buffer (KHB; 37°C) containing the following: 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl², 1.2 mm KH²PO⁴, 1.2 mm CaCl², 37.5 mm NaHCO³, and 16.5 mm dextrose, pH 7.35. After a 5-min equilibration period, the perfusion buffer was changed to Ca²⁺-free KHB containing collagenase type II (347 U/ml; Worthington Biochemical Corp., Freehold, NJ). After digestion with collagenase (20 min), the ventricles were minced and shaken in KHB, and the resulting cellular digest was washed, filtered, and resuspended in phosphate-free HEPES-buffered saline (HBS; 23°C) containing the following: 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl², 1.25 mm CaCl², 11.0 mm dextrose, 25.0 mm HEPES, and 5.0 mm pyruvate, pH 7.35.

Ventricular myocytes were also obtained from adult male Hartley guinea pigs as previously described. 19Briefly, excised hearts were subjected to coronary perfusion via  the aorta with KHB containing 120 mm NaCl, 4.8 mm KCl, 1.5 mm CaCl², 2.2 mm MgSO⁴, 1.2 mm NaH²PO⁴, 25 mm NaHCO³, and 11 mm glucose. The buffer's pH was maintained at 7.35 by bubbling with 95% O²–5% CO²at 37°C. Immediately after removal, the heart was perfused with normal Ca²⁺-containing KHB for 5 min, followed by Ca²⁺-free KHB for an additional 5 min, and then a Ca²⁺-free KHB containing collagenase B (0.5–0.7 mg/ml; Boehringer Mannheim, Indianapolis, IN) for 45 min. After perfusion, the atria were removed and the ventricles minced, rinsed free of collagenase, and reintroduced to Ca²⁺-containing KHB. Gentle trituration freed individual cells for use in patch clamp experiments.

Simultaneous measurement of [Ca²⁺]ⁱand cell shortening was performed as previously described. 7Ventricular myocytes (0.5 × 10⁶cells/ml) were incubated in HBS containing 2 μm fura-2–acetoxy methylester at room temperature for 15 min. Fura-2–loaded ventricular myocytes were placed in a temperature-regulated (28°C) chamber (Bioptechs, Inc., Butler, PA) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope (Olympus America, Lake Success, NY). The cells were superfused continuously with HBS at a flow rate of 2 ml/min and field-stimulated via  bipolar platinum electrodes at a frequency of 0.3 Hz with a 5-ms pulse using a Grass SD9 stimulator (Grass Instruments, West Warwick, RI). Myocytes showing a rod-shaped appearance with clear striations were chosen for study.

Fluorescence measurements were performed on individual myocytes using a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, Lawrenceville, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Because calibration procedures rely on a number of assumptions, the ratio of the light intensities at the two wavelengths was used to measure qualitative changes in [Ca²⁺]ⁱ. Just before data acquisition, background fluorescence was measured and automatically subtracted from the subsequent experimental measurement. The fluorescence sampling frequency was 100 Hz, and data were collected using software from Photon Technology International.

To simultaneously monitor cell shortening, the cells were also illuminated with red light. A dichroic mirror (600-nm cutoff) in the emission path deflected the cell image through a charge-coupled device video camera (Phillips VC 62505T; Marshall Electronics, Culver City, CA) into a video-edge detector (Crescent Electronics, Sandy, UT) with 16-ms resolution. The video-edge detector was calibrated using a stage micrometer so that cell lengths during shortening and relengthening could be measured.

The following parameters were calculated for each individual contraction: diastolic [Ca²⁺]ⁱand cell length, systolic [Ca²⁺]ⁱand cell length, change in [Ca²⁺]ⁱ(systolic [Ca²⁺]ⁱminus diastolic [Ca²⁺]ⁱ) and twitch amplitude, time to peak (Tp) for [Ca²⁺]ⁱand peak shortening, and time to 50% and 90% (T50r and T90r) diastolic [Ca²⁺]ⁱand 50% and 90% relengthening. Parameters from 10 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.

Microelectrodes were pulled from borosilicate glass capillary tubing (Corning 8161; Warner Instrument Corp., Hamden, CT) and had resistances between 0.5 and 1.5 MΩ when filled with the following intracellular solution: 125 mm CsCl, 20 mm TEA-Cl, 10 mm HEPES, 10 mm EGTA, 5 mm MgATP, and 3.6 mm creatine phosphate, pH 7.2. The control extracellular solution contained 140 mm NaCl, 5.4 mm CsCl, 2.5 mm CaCl², 0.5 mm MgCl², 5.5 mm HEPES, and 11 mm glucose; pH was adjusted to 7.4 with NaOH. Because of technical difficulties in obtaining a good seal with the patch pipette lasting the entire duration of the protocol, electrophysiologic studies in rat cardiomyocytes were not successful. Myocytes isolated from guinea pig hearts were placed in a 0.5-ml chamber (28°C) and superfused by gravity with experimental solutions. Currents were recorded with an Axopatch 200B voltage clamp amplifier (Axon Instruments, Union City, CA) and an IBM-compatible computer using pCLAMP software (Axon Instruments). A 3 m KCl-agar bridge was used to ground the bath. Cells were voltage clamped at −40 mV to inactivate the Na⁺current. The time course of changes in Ca²⁺conductance was monitored by applying a 75-ms test pulse to 0 mV once every 10 s. The magnitude of I^Cawas determined by measuring the peak inward current recorded during the step to 0 mV. The peak amplitude of I^Cawas normalized for cell membrane capacitance and the current density (in picoamperes per picofarad) was used to assess the effects of propofol. When the pulses were applied at 0.1 Hz, the rundown of I^Cain most myocytes occurred during the initial 5–10 min after the patch was broken. To minimize the influence of rundown on the measurements and to optimize comparisons between groups, the time window between 10–20 min after the initial recording was chosen to measure I^Cawith respect to drug effects. Only one concentration of drug was tested in each preparation. In addition, only currents that returned to at least 80% of their initial magnitude after washout were included in the study. Myocytes that showed marked or progressive rundown were excluded from the study.

Suspensions of rat ventricular myocytes were used for determining cAMP production. The experimental buffer was the same as that used in the [Ca²⁺]ⁱand shortening experiments (HBS). At the end of the protocol, the cells were quickly pelleted using a microfuge (500 g , 5 s). The buffer was aspirated and the pellet was resuspended in ice-cold HBS and centrifuged again (500 g , 5 s). The supernatant was aspirated and frozen using liquid nitrogen. Samples were then thawed on ice. Freezing and thawing was repeated three times. The preparations were homogenized in ice-cold ethanol (0.5 ml) to extract the cAMP. Homogenates were centrifuged (1,500 g , 10 min) and the supernatants collected. The pellet was washed with 0.5 ml ethanol-water (2:1) and centrifuged again at 1,500 g  for 10 min. The supernatants were combined and dried under nitrogen. Samples were stored at −20°C. Production of cAMP was assessed using an enzyme-linked immunoassay kit (Cayman Chemical, Ann Arbor, MI) and normalized for protein content using the method of Bradford. 20 

To determine the effect of propofol on steady state myocyte function, we examined changes in shortening and [Ca²⁺]ⁱduring exposure to propofol. Pure propofol was used to avoid any possible effect of the intralipid emulsion diluent on cell signaling pathways. Baseline measurements were collected from individual myocytes for 2 min in the absence of propofol. Myocytes were then exposed to propofol (1, 10, 30, and 100 μm) in a cumulative fashion and allowed to equilibrate for 5 min at each concentration.

To identify the extent to which propofol alters β-adrenoreceptor–mediated increases in [Ca²⁺]ⁱand shortening, we activated β adrenoreceptors with isoproterenol and examined the changes in myocyte shortening and [Ca²⁺]ⁱduring subsequent exposure to propofol. Baseline measurements were collected from individual myocytes for 2 min. Myocytes were exposed to isoproterenol (10 nm), and data were acquired after a 5-min equilibration period. Propofol (1, 10, 30, and 100 μm) was then added cumulatively to the isoproterenol-containing buffer and allowed to equilibrate. Data were acquired after a 5-min equilibration period. In other experiments, multiple doses of isoproterenol were used to assess the effects of propofol (3, 10, 30, or 100 μm) on the isoproterenol dose–response relation. When examining the role of PKC as a mediator of propofol-induced effects on isoproterenol-stimulated [Ca²⁺]ⁱand shortening, we pretreated the cells with the broad-range PKC inhibitor bisindolylmaleimide I (1 μm) 21or Gö 6976 (1 μm), an inhibitor of Ca²⁺-dependent PKC isoforms, 22for 10 min before stimulation with isoproterenol and incubation with propofol (1, 10, 30, 100 μm). Neither inhibitor had an effect on steady state or isoproterenol-stimulated [Ca²⁺]ⁱor shortening. When examining the specificity of propofol for the β-adrenergic signaling pathway, we increased cardiac inotropy using Bay K8644 (1 μm: L-type Ca²⁺channel agonist) and then added propofol cumulatively to the Bay K8644–containing buffer. Similar experiments were performed using forskolin (adenylyl cyclase activator) to bypass the β-adrenergic receptor and increase cardiac inotropy.

To determine whether propofol alters the β-adrenoreceptor–mediated increase in [Ca²⁺]ⁱand shortening via  inhibition of I^Ca, we measured the extent to which propofol alters both steady state I^Caand the isoproterenol-induced increase in I^Causing conventional whole cell patch clamp analysis. After rundown of the current reached a steady state (approximately 10 min), propofol (0.1, 1, 10 μm) or isoproterenol (30 nm) was added to the perfusate, and effects on I^Cawere assessed. In other experiments, the response to isoproterenol in the presence of propofol was measured. For these experiments, the control response to isoproterenol was first measured, followed by washout and pretreatment with propofol (10 min) before readdition of isoproterenol to the perfusate. In this fashion, each cell served as its own control. Ascorbic acid (50 μm) was added to all solutions to prevent oxidative degradation of isoproterenol.

Suspensions of rat ventricular myocytes (10⁶cells/ml) were preincubated with 3-isobutyl-1-methylxanthine (0.5 mm; phosphodiesterase inhibitor) for 5 min at 37°C. After 3-isobutyl-1-methylxanthine pretreatment, propofol (1, 10, 100 μm), isoproterenol (100 nm), or forskolin (1 μm) was added to the cells (10 min), and effects on steady state cAMP concentrations were assessed. In other experiments, propofol (0.1–100 μm) was added for 10 min before addition of isoproterenol (100 nm) or forskolin (1 μm). When examining the role of PKC as a mediator of propofol-induced effects on cAMP production, we pretreated the cells with bisindolylmaleimide I (1 μm) or Gö 6976 (1 μm) for 10 min before incubation with propofol (10 min) and stimulation with isoproterenol. Separate experiments included a positive control for PKC activation using phorbol myristate acetate (1 μm).

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 all hearts were weighted equally. Data are reported as mean ± standard error of the mean. Statistical comparisons within groups were made by one-way analysis of variance for repeated measures coupled with Student t  test. Two-way analysis of variance was used for between-group comparisons. A P  value < 0.05 was considered statistically significant.

Propofol, phorbol myristate acetate, and bisindolylmaleimide I were obtained from Research Biochemicals International (Natick, MA) and solubilized in dimethylsulfoxide to appropriate stock concentrations. Isoproterenol, forskolin, ascorbic acid, and 3-isobutyl-1-methyl-xanthine were obtained from Sigma Chemical Co. (St. Louis, MO). Gö 6976 was obtained from Calbiochem (La Jolla, CA).

The baseline 340/380 ratio was 0.87 ± 0.02. The change in the 340/380 ratio with shortening was 0.47 ± 0.03. Diastolic cell length was 113 ± 4 μm. Twitch height was 10.5 ± 2.3 μm (9.3 ± 1.5% resting cell length). Tp for [Ca²⁺]ⁱand shortening was 203 ± 5 and 245 ± 12 ms, respectively. T50r for [Ca²⁺]ⁱand shortening was 197 ± 8 and 224 ± 21 ms, respectively. T90r for [Ca²⁺]ⁱand shortening was 607 ± 52 and 579 ± 65 ms, respectively.

A representative trace depicting the dose-dependent effects of propofol on shortening and [Ca²⁺]ⁱin an electrically stimulated ventricular myocyte is shown in figure 1A. As we previously reported, 7low doses (1–30 μm) of propofol had no effect on shortening or peak [Ca²⁺]ⁱ. However, propofol (30 μm) reduced (P < 0.05) resting cell length from 113 ± 4 to 110 ± 3 μm with no effect on baseline [Ca²⁺]ⁱ. A supraclinical concentration of propofol (100 μm) attenuated shortening and peak [Ca²⁺]ⁱ. Summarized data for the dose-dependent effects of propofol on shortening and [Ca²⁺]ⁱare shown in figure 1B. An exploded view of the individual [Ca²⁺]ⁱtransient and shortening is illustrated in figure 2A, and continuous [Ca²⁺]ⁱ:cell length relations are depicted as hysteresis loops in figure 2B. Propofol (10 μm) had no significant effect on peak [Ca²⁺]ⁱor shortening (fig. 2A, left) but caused a slight upward shift in the continuous [Ca²⁺]ⁱ:shortening relation (fig. 2B). Figure 2A(right) is an overlay of the individual cell shortening and [Ca²⁺]ⁱtransient normalized to peak height to illustrate changes in timing. Propofol (10 μm) had no effect on the timing parameters (Tp, T50r, T90r) for either shortening or [Ca²⁺]ⁱunder steady state conditions.

A representative trace depicting cell shortening and [Ca²⁺]ⁱbefore and after exposure to isoproterenol is illustrated in figure 3A. During baseline conditions, the myocyte shortens from 120 μm down to 110 μm during contraction. After stimulation with isoproterenol (10 nm), cell shortening is increased, as demonstrated by cell length changing from 120 to 100 μm (223 ± 25%) during contraction that lasted up to 30 min. Isoproterenol also increased [Ca²⁺]ⁱ(147 ± 9%). The effects of isoproterenol on [Ca²⁺]ⁱand shortening were dose-dependent (fig. 3B). An exploded view of the individual [Ca²⁺]ⁱtransient and cell shortening is illustrated in figure 4A. Isoproterenol reduced (P < 0.05) Tp for shortening from 245 ± 9 to 203 ± 4 ms without any effect on Tp for [Ca²⁺]ⁱ. Isoproterenol (10 nm) reduced T50r and T90r for [Ca²⁺]ⁱfrom 197 ± 8 to 141 ± 5 ms and from 607 ± 52 to 313 ± 16 ms, respectively (P < 0.05). Isoproterenol also reduced T50r and T90r for shortening from 224 ± 21 to 149 ± 8 ms and from 579 ± 65 to 267 ± 9 ms, respectively (P < 0.05). In addition to this positive lusitropic effect, isoproterenol caused a rightward and upward shift in the [Ca²⁺]ⁱ:cell length relation (fig. 4B).

Figure 5Ais a representative trace depicting the dose-dependent effects of propofol on shortening and [Ca²⁺]ⁱafter exposure to isoproterenol (10 nm). The summarized data are shown in figure 5B. Propofol attenuated isoproterenol-induced increases in shortening and [Ca²⁺]ⁱin a dose-dependent manner. Pretreatment with propofol also caused dose-dependent decreases in isoproterenol-stimulated changes in shortening and [Ca²⁺]ⁱ(fig. 3B). Figure 6A(left) is an exploded view of the individual [Ca²⁺]ⁱtransients and cell shortening, demonstrating attenuation by propofol (10 μm) of the isoproterenol-induced positive inotropic effect. Figure 6A(right) illustrates that the lusitropic effect of isoproterenol on shortening was not altered by propofol. Propofol (10 μm) partially reversed the effect of isoproterenol on T50r and T90r for [Ca²⁺]ⁱfrom 142 ± 15 to 168 ± 12 ms (P < 0.08) and from 322 ± 12 to 388 ± 9 ms (P < 0.07), respectively. Propofol (100 μm) prolonged (P < 0.05) T50r and T90r for [Ca²⁺]ⁱfrom 137 ± 8 to 185 ± 14 ms and 312 ± 15 to 427 ± 11 ms, respectively. There was no concomitant effect of propofol (100 μm) on T50r or T90r for shortening. Figure 6Bshows the continuous [Ca²⁺]ⁱ:shortening relation. Propofol attenuated the rightward and upward shift in the hysteresis loop induced by isoproterenol. In control experiments, propofol (30 μm) did not alter Bay K8644–induced increases in [Ca²⁺]ⁱ(92 ± 7% of control) or shortening (95 ± 8% of control).

The effect of propofol on steady state I^Cais shown in figure 7A. Propofol did not alter steady state I^Ca. The effect of propofol on isoproterenol-stimulated increases in I^Cais shown in figure 7B. Isoproterenol (30 nm) increased peak I^Caby 123 ± 13% compared with the steady state control. Propofol reduced the isoproterenol-stimulated increase in I^Cain a dose-dependent manner.

Propofol alone did not alter basal cAMP accumulation (fig. 8A). Isoproterenol (100 nm) increased cAMP production by 614 ± 37%. Pretreatment with propofol caused a dose-dependent reduction in the isoproterenol-stimulated increase in cAMP (fig. 8B). In contrast, direct activation of adenylyl cyclase with forskolin (1 μm) stimulated a 494 ± 70% increase in cAMP production that was not altered by pretreatment with propofol (fig. 8C). Propofol (30 μm) also did not alter forskolin-induced increases in [Ca²⁺]ⁱ(93 ± 7% of control) or shortening (94 ± 9% of control).

Figure 9Ademonstrates that PKC activation with phorbol myristate acetate caused a dose-dependent decrease in isoproterenol-stimulated cAMP production. Inhibition of PKC with the broad-range inhibitor bisindolylmaleimide I (1 μm) prevented the propofol-induced decrease in isoproterenol-stimulated cAMP production (fig. 9B). Similarly, inhibition of Ca²⁺-dependent PKC isoforms with Gö 6976 abolished the propofol-induced decrease in isoproterenol-stimulated cAMP production (fig. 9C).

Figure 10Ademonstrates that PKC inhibition with bisindolylmaleimide I abolished the propofol-induced reduction of isoproterenol-stimulated [Ca²⁺]ⁱand shortening at clinically relevant concentrations. Inhibition of Ca²⁺-dependent PKC isoforms with Gö 6976 had similar effects on propofol-induced changes in isoproterenol-stimulated [Ca²⁺]ⁱand shortening (Fig. 10B).

Because neural, humoral, and local mechanisms interact to regulate myocardial contractility in vivo , in vitro  preparations allow for assessment of direct actions of anesthetics on cellular mechanisms that regulate cardiac function. To our knowledge, this is the first study to directly measure [Ca²⁺]ⁱ, shortening, I^Ca, and cAMP production in cardiomyocytes during β-adrenoreceptor stimulation in the presence or absence of propofol. Our major findings are that propofol, at clinically relevant concentrations, has no effect on steady state [Ca²⁺]ⁱ, shortening, I^Ca, or cAMP levels. In contrast, propofol attenuates isoproterenol-stimulated increases in [Ca²⁺]ⁱ, shortening, I^Ca, and cAMP production. The inhibitory site of action within the β-adrenergic signal transduction pathway is upstream of adenylyl cyclase and involves activation of a PKC-dependent pathway.

The effects of propofol on myocardial contractility have previously been evaluated using a variety of experimental preparations. Results from most in vitro  studies indicate that propofol exerts little if any direct negative inotropic effect at clinically relevant concentrations. 4,7,11However, propofol is known to have multiple sites of action, including inhibitory effects on L-type Ca²⁺channels 23–25and sarcoplasmic reticulum Ca²⁺handling. 26These inhibitory effects on Ca²⁺dynamics would result in negative inotropy, but cardiac depression could be masked by a concomitant increase in myofilament Ca²⁺sensitivity 7,27,28Our results confirm previous findings by our laboratory using the intralipid emulsion form of propofol 7and demonstrate that pure propofol exerts no significant inhibitory effect on steady state [Ca²⁺]ⁱor shortening at clinically relevant concentrations. Similar to our previous findings, 7supraclinical concentrations of propofol (100 μm) decreased peak [Ca²⁺]ⁱand shortening, likely via  inhibitory effects on the I^Ca. 23,24Propofol also caused a decrease in resting cell length with no concomitant increase in baseline [Ca²⁺]ⁱ, indirectly confirming that propofol increases myofilament Ca²⁺sensitivity. 27,28 

Activation of β adrenoreceptors by catecholamines is an important mechanism for increasing the inotropic state of the heart. In this study, activation of β adrenoreceptors with isoproterenol resulted in positive inotropy and lusitropy, as previously reported by other investigators. 12,29The response to β-adrenoreceptor stimulation is mediated via  a G-protein–dependent increase in adenylyl cyclase activity and cAMP production within the myocyte, causing activation of protein kinase A. The positive inotropic effect is thought to be primarily mediated by protein kinase A–dependent phosphorylation of the L-type Ca²⁺channel, resulting in an increase in I^Caand [Ca²⁺]ⁱ. 16In contrast, the positive lusitropic effect is thought to be mediated primarily by protein kinase A–dependent changes in troponin I phosphorylation, causing decreased affinity of troponin C to bind Ca²⁺(i.e. , a decrease in myofilament Ca²⁺sensitivity). 17Protein kinase A–dependent phosphorylation of phospholamban enhances the rate of uptake of Ca²⁺into the sarcoplasmic reticulum during relaxation and increases the amount of Ca²⁺available for release during contraction, thereby contributing to both lusitropic and inotropic effects, respectively. 18Therefore, activation of β adrenoreceptors induces important effects on myocardial contraction and relaxation that are mediated via  an increase in [Ca²⁺]ⁱand a concomitant decrease in myofilament Ca²⁺sensitivity.

Propofol is widely used for the sedation of critically ill patients who are receiving catecholamines for hemodynamic support. However, the extent to which propofol alters β-adrenoreceptor signal transduction in cardiac muscle remains controversial. Hebbar et al.  13,14reported that clinically relevant concentrations of propofol (1 μg/ml = 5.6 μm) reduced the inotropic response of swine cardiomyocytes to isoproterenol, although the site of action and cellular mechanism were not investigated. Propofol has been reported to reduce myocardial β-adrenoreceptor ligand binding and responsiveness in rat myocardium. 15In contrast, Lejay et al.  12reported that propofol (10 μg/ml = 56 μm) did not alter the isoproterenol-induced positive inotropic effect but enhanced the positive lusitropic effect in rat papillary muscle. In our study, propofol (10 μm) attenuated the isoproterenol-induced increase in [Ca²⁺]ⁱand shortening, which indicates that propofol inhibits β-adrenoreceptor–mediated cardiac inotropy at clinically relevant concentrations. This was not a nonspecific effect, because propofol did not inhibit increases in [Ca²⁺]ⁱand shortening in response to the L-type Ca²⁺channel agonist, Bay K8644. The lusitropic effect of isoproterenol was partially reversed by propofol at low concentrations but only reached statistical significance at 100 μm. Because the lusitropic effect is primarily mediated via  alterations in the binding affinity of troponin C for Ca²⁺(decrease Ca²⁺sensitivity), it is possible that the partial reversal of the isoproterenol-induced lusitropic effect by propofol could be mediated by an increase in the affinity of troponin C for Ca²⁺(increased Ca²⁺sensitivity). This would be consistent with our previous study demonstrating a propofol-induced increase in myofilament Ca²⁺sensitivity mediated by a Na⁺–H⁺exchange-dependent increase in intracellular pH. 28 

The increase in [Ca²⁺]ⁱafter β-adrenoreceptor activation is mediated primarily via  an increase in the I^Ca. Some studies have indicated that propofol can directly inhibit I^Cain cardiomyocytes, 23–25perhaps through a direct interaction with the dihydropyridine-binding site, 24although the clinical relevance is still controversial. 25In our study, clinically relevant doses of propofol (0.1–10 μm) had no significant effect on steady state I^Cabut attenuated the increase in I^Cainduced by isoproterenol. These data support the concept that the propofol-induced depression of the isoproterenol-stimulated increase in [Ca²⁺]ⁱand shortening is mediated by a decrease in I^Ca, although we cannot rule out an effect of propofol on sarcoplasmic reticulum Ca²⁺stores. 26,27Again, these data further support the hypothesis that propofol interferes with the β-adrenergic signaling pathway in cardiomyocytes.

If propofol is directly interacting with the β-adrenoreceptor signaling pathway, then propofol should attenuate isoproterenol-induced increases in cAMP production. Propofol had no effect on steady state cAMP concentrations in cardiomyocytes. However, propofol attenuated the isoproterenol-induced increase in cAMP production in a dose-dependent manner. In contrast, propofol did not alter the increase in cAMP induced by direct activation of adenylyl cyclase with forskolin. These data indicate that propofol interacts with the β-adrenoreceptor signaling pathway at a site upstream of adenylyl cyclase. Other intravenous anesthetics have been reported to alter cAMP production in response to agonist activation. Ketamine inhibited cytokine-induced reductions in intracellular cAMP accumulation in a rat heart cell line, 30and diazepam enhanced cAMP production induced by isoproterenol and forskolin. 31 

We recently reported that propofol increases myofilament Ca²⁺sensitivity and intracellular pH via  a PKC-dependent activation of the Na⁺–H⁺exchanger. 28In addition, it has been demonstrated that activation of PKC attenuates β-adrenergic responsiveness in the rat heart 32and β-adrenergic–mediated increases in I^Cain rat ventricular myocytes. 33In the current study, phorbol myristate acetate attenuated isoproterenol-induced cAMP production. This indicates that PKC activation can negatively regulate β-adrenoreceptor signal transduction, resulting in a decrease in cAMP production. In addition, the propofol-induced inhibition of isoproterenol-stimulated cAMP production was attenuated by bisindolylmaleimide I and Gö 6976. Furthermore, bisindolylmaleimide I and Gö 6976 virtually abolished the propofol-induced inhibition of isoproterenol-stimulated increases in [Ca²⁺]ⁱand shortening, which further supports a functional role for PKC in mediating the effects of propofol. Taken together, these data suggest that the mechanism by which propofol exerts its effects on the β-adrenergic signaling pathway is via  activation of a Ca²⁺-dependent PKC isoform. The isoform involved is likely PKCα, because this is the only Ca²⁺-dependent PKC isoform that exists in adult rat cardiomyocytes, 34and its site of action appears to be upstream of adenylyl cyclase. One possibility is that propofol-induced activation of PKC may result in direct phosphorylation of the β-adrenergic receptor, leading to receptor desensitization. 35Alternatively, PKC activation may indirectly cause β-adrenergic receptor desensitization via  phosphorylation of β-adrenergic receptor kinases. 36,37 

One limitation of the study is that the experiments were performed at 28°C with a low stimulation rate. The studies were performed during these conditions to maintain myocyte viability and reduce spontaneous contractions. These experimental conditions could alter excitation–contraction coupling or enzymatic regulatory processes compared with in vivo  conditions. Another potential limitation is that pure propofol solubilized in dimethylsulfoxide was used instead of the commercially available 10% intralipid emulsion. This allowed us to directly assess the effects of propofol, independent of the vehicle, on cardiac inotropy. In addition, guinea pig myocytes were used for the electrophysiologic studies. It is known that there are differences between rat and guinea pig myocytes in terms of action potential characteristics, sarcoplasmic reticulum Ca²⁺handling, and the Na⁺–Ca²⁺exchanger in the steady state regulation of excitation contraction coupling. Finally, the validity of interpreting externally unloaded myocyte shortening as an indicator of the inotropic state of the myocardium is a potential limitation, because the force developed during contraction is unknown. Even without an external load, the myocytes are shortening against an internal load composed of several components. 38 

In summary, propofol, at clinically relevant concentrations, attenuates β-adrenergic signal transduction in cardiac myocytes via  inhibition of cAMP production. The inhibitory site of action of propofol appears to be upstream of adenylyl cyclase and involves activation of PKC.