Propofol Increases Contractility during α^1a-Adrenoreceptor Activation in Adult Rat Cardiomyocytes

All experimental procedures and protocols were approved by the Cleveland Clinic Foundation Institutional Animal Care and Use Committee (Cleveland, Ohio) and conform to the international guidelines for the care and use of animals.

Ventricular myocytes were freshly isolated from adult male Sprague-Dawley rat hearts as previously described.15,16Immediately after euthanasia, the hearts were rapidly removed and perfused in a retrograde manner at a constant flow rate (8 ml/min) with oxygenated (95% oxygen–5% carbon dioxide) Krebs-Henseleit buffer (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, at a pH of 7.35. After a 5-min equilibration period, the perfusion buffer was changed to Ca²⁺-free Krebs-Henseleit buffer 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 Krebs-Henseleit buffer, and the resulting cellular digest was washed, filtered, and resuspended in phosphate-free HEPES-buffered saline (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, at a pH of 7.35.

Simultaneous measurement of [Ca²⁺]ⁱand myocyte shortening was performed as previously described.15,16Ventricular myocytes were incubated in HEPES-buffered saline containing 2 μm fura-2/AM at 37°C for 20 min. Fura-2–loaded ventricular myocytes were placed in a temperature-regulated chamber (Bioptechs Inc., Butler, PA) mounted on the stage of an Olympus IX70 inverted fluorescence microscope (Olympus America, Lake Success, NY). The cells were superfused continuously with HEPES-buffered saline throughout the experiment and field-stimulated via  bipolar platinum electrodes using a Grass SD9 stimulator (Grass Instruments, West Warwick, RI). Fluorescence measurements were performed on single ventricular myocytes using a dual-wavelength spectrofluorometer 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 above 600 nm for simultaneous measurement of myocyte shortening using a video-edge detector (Crescent Electronics, Sandy, UT). The video-edge detector was calibrated using a stage micrometer so that cell lengths during shortening and relengthening could be measured. 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 a software package from Photon Technology International (Lawrenceville, NJ). Fluorescence data for the [Ca²⁺]ⁱtransients and myocyte shortening were imported into Lab View (National Instruments, Austin TX), where both the [Ca²⁺]ⁱtransients and myocyte contractile responses were analyzed synchronously and simultaneously. We verified that propofol had no effect on fura-2 fluorescence at the concentrations tested. This was confirmed in separate cell-free experiments using fura-2 (pentapotassium salt) in buffers ranging from 10⁻⁹m to 10⁻⁵m in the presence or absence of propofol (data not shown).

The following variables were calculated for each individual contraction: resting [Ca²⁺]ⁱand cell length; peak [Ca²⁺]ⁱand cell length; change in [Ca²⁺]ⁱ(peak [Ca²⁺]ⁱminus resting [Ca²⁺]ⁱ) and twitch amplitude; time to peak (Tp) for [Ca²⁺]ⁱand shortening and time to 50% (Tr) resting [Ca²⁺]ⁱand relengthening. Variables from 10 contractions were averaged to obtain mean values at baseline and in response to the various interventions. Averaging the variables over time minimizes beat-to-beat variation.

Baseline measurements were collected from individual myocytes for 5 min in the presence of chloroethylclonidine (1 μm), an inhibitor of α^1b-AR, and BMY 7378 (1 μm), an inhibitor of α^1d-AR, in this and all subsequent protocols. Alpha-1a ARs were subsequently activated with phenylephrine (1–100 μm), and [Ca²⁺]ⁱand shortening were measured until a new steady state was achieved (10 min). The new steady state values in the presence of phenylephrine are referred to as the control responses.

Phenylephrine (10 μm) was applied to the cardiomyocyte, and [Ca²⁺]ⁱand shortening were monitored. After establishment of a new steady state increase in shortening, phenylephrine was washed out (10 min), and U73122 (50 μm; IC⁵⁰= 2 μm) or AACOCF³(50 μm; IC⁵⁰= 15 μm) was added (10 min) to inhibit phospholipase C (PLC) or phospholipase A²(PLA²), respectively. Phenylephrine (10 μm) was again added to the cardiomyocyte until the increase in shortening had achieved a new steady state. Time control experiments were also performed in the absence of the inhibitor. This approach was used in all protocols assessing the effects of putative inhibitors on the phenylephrine response.

Cardiomyocytes were pretreated (10 min) with the broad-range PKC inhibitor bisindolylmaleimide I (Bis, 10 μm; IC⁵⁰= 10 nm) or the ROK inhibitor Y27632 (10 μm; IC⁵⁰= 140 nm), and the changes in myocyte shortening and [Ca²⁺]ⁱwere examined during subsequent exposure to phenylephrine (10 μm).

Alpha-1a ARs were activated with phenylephrine (10 μm) in individual cardiomyocytes, and the changes in myocyte [Ca²⁺]ⁱand shortening were examined during subsequent exposure to propofol (1, 10, 30 μm).

Cardiomyocytes were pretreated with the broad-range PKC inhibitor Bis (10 μm); a selective inhibitor of Ca²⁺-dependent PKC isoforms, Gö 6976 (10 μm; IC⁵⁰= 2 nm); a selective inhibitor of PKCδ, rottlerin (10 μm; IC⁵⁰= 3 μm); a selective inhibitor of PKCϵ, myristoylated PKCϵ V1-2 (10 μm; IC⁵⁰= 100 nm); or a selective inhibitor of PKCζ, myristoylated PKCζ inhibitor peptide (10 μm; IC⁵⁰= 100 nm) for 10 min after stimulation with phenylephrine (10 μm) and before incubation with propofol (1, 10, 100 μm). In a separate set of experiments, isoproterenol (1 μm) was added to demonstrate that a ceiling effect on cardiomyocyte shortening had not been achieved in the presence of phenylephrine and Bis.

Activation of Na⁺–H⁺exchange results in intracellular alkalinization, which increases myofilament Ca²⁺sensitivity,17and propofol increases intracellular pH in cardiomyocytes via  a PKC-dependent pathway.16Cardiomyocytes were pretreated (10 min) with or without the Na⁺–H⁺exchange inhibitor HOE 694 (1 μm; IC⁵⁰= 50 nm) before addition of propofol.

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. Comparison of several means was performed using two-way analysis of variance and the Newman-Keuls test. The Bonferroni post hoc  correction was used when significant differences among the groups were detected. All P  values are one tailed, and differences were considered significant at P < 0.05. All results are expressed as mean ± SD.

Propofol was obtained from Aldrich Chemical Co. (Milwaukee, WI). Phenylephrine, chloroethylclonidine, and BMY 7378 were obtained from Sigma Chemical Co. (St. Louis, MO). Phorbol myristic acetate, HOE 694, Bis, and Y-27632 were obtained from Calbiochem (San Diego, CA). AACOCF³, U73122, Gö 6976, myristoylated PKCϵ V1-2 peptide, myristoylated PKCζ inhibitor peptide, and rottlerin were obtained from BIOMOL (Plymouth Meeting, PA). Fura-2/AM was obtained from Texas Fluorescence Labs (Austin, TX).

Resting cell length was 132 ± 9 μm, and the baseline 340/380 ratio was 0.9 ± 0.2. Twitch height was 11.0 ± 1.8 μm (8.3 ± 1.4% of the resting cell length). The change in 340/380 ratio from baseline with shortening was 0.6 ± 0.1. Tp [Ca²⁺]ⁱand Tp shortening were 143 ± 18 and 171 ± 21 ms, respectively. Tr [Ca²⁺]ⁱand Tr shortening were 183 ± 18 and 217 ± 24 ms, respectively.

The combined presence of chloroethylclonidine (1 μm) and BMY 7378 (1 μm) had no effect on baseline values for [Ca²⁺]ⁱor shortening (data not shown). A representative trace depicting the effect of phenylephrine on [Ca²⁺]ⁱand shortening in a single field-stimulated ventricular myocyte is shown in figure 1A. Phenylephrine (10 μm) increased peak shortening without a concomitant increase in [Ca²⁺]ⁱ. A decrease in resting cell length of 2.0 ± 0.4 μm with no change in resting [Ca²⁺]ⁱwas observed in most cells. Summarized data for the concentration-dependent effects of phenylephrine on [Ca²⁺]ⁱand shortening are shown in figure 1B. Phenylephrine caused concentration-dependent increases in shortening (P = 0.001), with no significant change in [Ca²⁺]ⁱ. Phenylephrine (10 μm) had no significant effect on Tp [Ca²⁺]ⁱ(97 ± 6% of baseline; not significant [NS]), Tp shortening (95 ± 5% of baseline; NS), Tr [Ca²⁺]ⁱ(93 ± 8% of baseline; NS), or Tr shortening (92 ± 7% of baseline; NS).

Neither U73122 nor AACOCF³alone had an effect on baseline [Ca²⁺]ⁱor shortening. Summarized data for the effects of U73122 or AACOCF³on the phenylephrine-induced increase in shortening are depicted in figure 2. Inhibition of PLC with U73122 reduced the phenylephrine-induced increase in shortening by 15 ± 6% (P = 0.017), whereas inhibition of PLA²with AACOCF³attenuated the phenylephrine-induced increase in shortening by 84 ± 11% (P = 0.003).

Summarized data for the effects of Bis (10 μm) and Y-27632 (10 μm) on the phenylephrine-induced (10 μm) increase in shortening are shown in figure 3. Inhibition of PKC with Bis reduced the phenylephrine-induced increase in shortening by 17 ± 8% (P = 0.014), whereas inhibition of ROK with Y-27632 inhibited the phenylephrine-induced increase in shortening by 74 ± 13% (P = 0.001).

A representative trace depicting the effect of propofol (30 μm) on [Ca²⁺]ⁱand shortening during α^1a-AR activation with phenylephrine (10 μm) is shown in figure 4A. Summarized data depicting the concentration-dependent effects of propofol on [Ca²⁺]ⁱand shortening during α^1a-AR are shown in figure 4B. Propofol caused concentration-dependent increases in shortening (P = 0.002), with no significant effect on [Ca²⁺]ⁱ. Propofol (30 μm) had no significant effect on Tp [Ca²⁺]ⁱ(103 ± 7%; NS), Tp shortening (98 ± 5%; NS), Tr [Ca²⁺]ⁱ(104 ± 5%; NS), or Tr shortening (109 ± 5%; NS) during α^1a-AR activation.

A representative trace depicting the effect of PKC inhibition with Bis on the propofol-induced increase in shortening is shown in figure 5A. Summarized data for the concentration-dependent effects of propofol on [Ca²⁺]ⁱand shortening in the presence of Bis are shown in figure 5B. After α^1a-AR activation and in the continued presence of phenylephrine, addition of Bis (10 μm) increased [Ca²⁺]ⁱand shortening by 14 ± 4% (P = 0.021) and 27 ± 6% (P = 0.003), respectively. Under these conditions, Bis completely inhibited the propofol-induced increase in shortening. However, activation of the β-AR signaling pathway with isoproterenol (10 nm) after pretreatment with phenylephrine and Bis increased [Ca²⁺]ⁱand shortening by 25 ± 5% (P = 0.001) and 48 ± 9% (P = 0.001), respectively, which demonstrates that the inhibitory effect of Bis on propofol-induced increases in shortening is not due to a ceiling effect. Propofol (30 μm) had no effect on Tp [Ca²⁺]ⁱ(97 ± 6%; NS), Tp shortening (94 ± 8%; NS), Tr [Ca²⁺]ⁱ(101 ± 7%; NS), or Tr shortening (96 ± 6%; NS) in the presence of Bis or during α^1a-AR activation.

We next assessed the extent to which selective inhibitors of specific PKC isoforms were involved in mediating the propofol-induced increase in shortening. Summarized data for the effects of PKCα inhibition with Gö 6976 (10 μm), PKCδ inhibition with rottlerin (10 μm), PKCϵ inhibition with myristoylated PKCϵ V1-2 (10 μm), and PKCζ inhibition with myristoylated PKCζ inhibitor peptide (10 μm) on the propofol-induced increase in shortening are shown in figure 6. Inhibition of PKCα, PKCδ, PKCϵ, and PKCζ reduced the propofol-induced increase in shortening during α^1a-AR activation by 12 ± 5% (P = 0.011), 36 ± 8% (P = 0.001), 32 ± 9% (P = 0.007), and 19 ± 5% (P = 0.008), respectively.

The Na⁺–H⁺exchange inhibitor HOE 694 (10 μm) had no significant effect on cell shortening during α^1a-AR activation with phenylephrine (96 ± 8% of phenylephrine response; NS). Summarized data for the effects HOE 694 on the propofol-induced increase in shortening are shown in figure 7. HOE 694 attenuated the propofol-induced increase in shortening during α^1a-AR activation by 47 ± 9% (P = 0.001).

Although propofol is widely used for the sedation of critically ill patients who are receiving catecholamines for hemodynamic support, relatively little is known about the interaction of propofol with adrenoreceptor stimulation. There is only one previous study that demonstrated that propofol abolished the positive inotropic effect of phenylephrine in isolated rat ventricular papillary muscles but enhanced the positive inotropic effect of isoproterenol (β-AR activation).11We previously demonstrated that propofol attenuated β-AR–mediated increases in [Ca²⁺]ⁱand shortening via  a PKC-dependent pathway in cardiomyocytes, at a site upstream of adenylyl cyclase.10This is the first in vitro  study to directly assess the effects of propofol on [Ca²⁺]ⁱand contractility in isolated ventricular cardiomyocytes in the setting of α^1a-AR activation. The key findings of this study are that the positive inotropic effect of α^1a-AR activation is mediated primarily via  a ROK-dependent increase in myofilament Ca²⁺sensitivity. Moreover, in the setting of α^1a-AR activation, propofol increases cardiomyocyte shortening with no concomitant effect on [Ca²⁺]ⁱ, indicating a propofol-induced increase in myofilament Ca²⁺sensitivity. The increased sensitivity seems to involve PKC activation and Na⁺–H⁺exchange. Figure 8represents a schematic of the proposed signaling pathways and cellular mechanisms for α^1a-AR activation and propofol in cardiomyocytes.

It is well established that three pharmacologically distinct α-AR subtypes (α^1a, α^1b, and α^1d) exist in rat cardiomyocytes and in the human heart.5,18,19However, the signal transduction pathways associated with activation of individual α-AR subtype activation and functional consequences on myocardial contractility are still controversial, likely due to activation of multiple parallel signaling pathways by each subtype.5Two possible mechanisms have been proposed to explain the inotropic response to α-AR activation: an increase in myofilament Ca²⁺sensitivity or an increase in transsarcolemmal Ca²⁺influx.6,8,20One recent report indicated opposing effects of α¹-adrenergic subtypes (α^1avs. α^1b) on [Ca²⁺]ⁱ, intracellular pH, and contractility in rat cardiac myocytes.3Opposing effects of the α-AR subtypes on intracellular [Ca²⁺]ⁱand pH regulation may explain in part the observed increase in cardiomyocyte contractility independent of an increase in [Ca²⁺]ⁱin response to the α¹-AR selective agonist phenylephrine.21In our study, selective activation of α^1a-AR resulted in an increase in shortening with no concomitant increase in [Ca²⁺]ⁱ. These results indicate that the primary mechanism by which α^1a-AR activation increases cardiomyocyte shortening is via  an increase in myofilament Ca²⁺sensitivity.

Alpha-1 ARs have been shown to couple to a variety of cellular phospholipases, including PLC and PLA², resulting in production of several important second messengers capable of modulating [Ca²⁺]ⁱor myofilament Ca²⁺sensitivity or both, including diacylglycerol, inositol trisphosphate, arachidonic acid, and RhoA.4,5,12,13However, the extent to which α^1a-AR–mediated increases in cardiomyocyte contractility are induced by these second messengers has not been clearly defined. In our study, inhibition of PLC had a minimal effect on the α^1a-AR–mediated increase in cardiomyocyte shortening, whereas PLA²inhibition attenuated the response by more than 80%. These data indicate that the α^1a-AR–mediated increase in cardiomyocyte shortening may involve the release of arachidonic acid from membrane phospholipids and that diacylglycerol release and PKC activation play a minimal role. We previously demonstrated that exogenously added arachidonic acid attenuates the transient outward K⁺current in cardiomyocytes,22resulting in an increase in cardiomyocyte [Ca²⁺]ⁱand shortening.23The lack of an increase in [Ca²⁺]ⁱin this study may be due to intracellular release of arachidonic acid by PLA²compared with exogenously applied arachidonic acid in our previous study.22We also demonstrated an arachidonic acid–dependent increase in the phosphorylation of the contractile proteins troponin I and myosin light chain 2, which can modulate myofilament Ca²⁺sensitivity.24Our data are also consistent with previous studies indicating that α^1a-AR activation results in a positive inotropic effect independent of PLC activation and phosphoinositide hydrolysis.25–28 

We investigated the extent to which protein kinases play a role in mediating the α^1a-AR–induced increase in cardiomyocyte shortening. Previous studies have suggested that activation of PKC plays a role in mediating the inotropic response to α-AR activation.2,4,29However, more recent studies have suggested that the α-AR–induced positive inotropic effect is independent of PKC activation and is a result of myosin light chain phosphorylation mediated by myosin light chain kinase activation, activation of ROK, or both.12–14In the current study, PKC inhibition attenuated the α^1a-AR–mediated increase in cardiomyocyte shortening by less than 20%, whereas ROK inhibition resulted in greater than 70% inhibition of the response. These data suggest that both PKC and ROK are mediators of the α^1a-AR–induced increase in cardiomyocyte shortening, with ROK playing a predominant role. It is likely that diacylglycerol formation due to PLC activation and arachidonic acid production from PLA²activation play an important role in activating these kinases. Interestingly, diacylglycerol and arachidonic acid synergistically increase cardiomyocyte contraction via  activation of PKC.30The mechanism by which PKC activation or arachidonic acid release results in an increase in myofilament Ca²⁺sensitivity may involve a direct phosphorylation of myosin light chain 2,24,31,32or may be due to indirect phosphorylation via  inhibition of myosin light chain phosphatase, as previously described in smooth muscle.33,34Further experiments are required to identify the precise mechanisms.

Recent studies have implicated activation of RhoA, a member of the Rho family of small-molecular-weight guanosine 5′-triphosphate–binding proteins, in mediating α¹-adrenergic signaling in cardiomyocytes12and in failing hearts.14Direct activation of RhoA by Gα^qafter α^1a-AR activation,12G protein–coupled release of arachidonic acid as a consequence of PLA²activation,33,35or both are likely involved in mediating the observed increase in cardiomyocyte shortening observed in our study. The mechanism likely involves the activation of ROK by Gα^q, arachidonic acid, or both,35,36leading to an inhibition of the myosin light chain phosphatase resulting in an increase in myosin light chain phosphorylation.33It has been recently demonstrated that the α¹-AR–induced positive inotropic response in the heart is dependent on myosin light chain phosphorylation.13 

We previously demonstrated that propofol had no effect on [Ca²⁺]ⁱand contraction of individual myocyte at clinically relevant concentrations37but attenuated the inotropic response to β-AR activation in cardiomyocytes.10In the current study, propofol increased cardiomyocyte shortening with no concomitant effect on [Ca²⁺]ⁱduring activation of α^1aAR with phenylephrine. In contrast, a previous study indicated that propofol abolished the inotropic effect of phenylephrine in isolated papillary muscles.11There are several reasons that may explain the differences between the findings. The major difference between the two studies is that the current study examines the effects of propofol during α-AR activation, whereas the study by Lejay et al.  11examined the propofol-induced modification of the inotropic response to α-AR activation (pretreatment with propofol). Moreover, the previous study11did not isolate a single signaling pathway (α^1a, α^1b, and α^1dAR are all activated by phenylephrine, which can have opposing effects on cardiomyocyte shortening3), whereas the current study isolates the α^1a-AR signaling pathway. In addition, differences in the extracellular Ca²⁺concentration of the experimental buffers can affect the inotropic effects of propofol.38Finally, the isolated cardiomyocytes in the current study were not loaded, and therefore, the resting cell length and myofilament Ca²⁺sensitivity may be modified and may contribute to the differences observed between the two studies. Regardless, the results of the current study indicate that propofol increases myofilament Ca²⁺sensitivity during α^1a-AR activation. Our data are the first to directly demonstrate a positive inotropic effect of propofol on cardiomyocyte shortening during α^1a-AR activation mediated by an increase in myofilament Ca²⁺sensitivity.

The extent to which propofol-induced changes in cardiomyocyte signaling are mediated via  activation of PKC has been actively explored by our laboratory.10,15,16However, isozyme specificity for specific cellular interactions has not been extensively examined. In our study, inhibition of PKC with Bis during α^1aAR resulted in an increase in cardiomyocyte [Ca²⁺]ⁱand shortening. This may be explained by an inhibition in tonic activity of PKC isoforms involved in limiting the availability of [Ca²⁺]ⁱand hence cardiomyocyte shortening.39Alternatively, direct block of human ether-a-go-go –related gene potassium channels by Bis has recently been reported,40which results in action potential prolongation and would increase [Ca²⁺]ⁱand cardiomyocyte shortening. We observed that PKC inhibition with Bis prevented the propofol-induced increase in cardiomyocyte shortening after α^1a-AR activation. Moreover, PKCδ and PKCϵ seem to be the predominant isoforms involved in mediating the response, with PKCα and PKCζ playing a lesser role. However, it seems that all four PKC isoforms have some role in mediating the increase in cardiomyocyte shortening in response to propofol, because the sum of their inhibitory effects accounts for virtually all of the propofol effect. The inability of propofol to increase shortening after inhibition of PKC with Bis was not due to a ceiling effect on cardiomyocyte shortening, because activation of the β-AR signaling pathway further increased both [Ca²⁺]ⁱand shortening. It remains to be determined what specific roles each of the individual isoforms play in the propofol-induced increase in shortening. Activation of PKC has been shown to increase myofilament Ca²⁺sensitivity via  phosphorylation of contractile proteins,15changes in intracellular pH, or both.16Little is known about the relative roles of the PKC isoforms in regulating myofibrillar protein phosphorylation,41,42and even less is known about isoform-specific modulation of Na⁺–H⁺exchange.43 

To identify a cellular mechanism to explain the propofol-induced increase in cardiomyocyte shortening, we examined whether Na⁺–H⁺exchange inhibition attenuates the propofol-induced increase in shortening after α^1a-AR activation. Our results indicate that Na⁺–H⁺exchange inhibition had little effect on shortening during α^1a-AR activation, indicating that intracellular alkalinization, which can increase myofilament Ca²⁺sensitivity, was not likely the mechanism for the increase in shortening in response to α^1a-AR activation. In contrast, Na⁺–H⁺exchange inhibition attenuated the propofol-induced increase in shortening during α^1a-AR activation, indicating a propofol-induced activation of Na⁺–H⁺exchange. These data suggest that intracellular alkalinization plays a role in mediating the propofol-induced increase in cardiomyocyte shortening during α^1a-AR activation.

Extrapolation of results obtained from in vitro  studies at the cellular level to the clinical setting can be difficult. However, it is known that PKC activation is a key mediator of ischemic and anesthetic preconditioning and cardiac protection. In addition, increases in myofilament Ca²⁺sensitivity can partially offset the negative inotropic effects of certain agents, including propofol. Therefore, propofol may be beneficial in patients exhibiting end-stage heart failure (or other cardiomyopathies) where Ca²⁺overload is observed. Moreover, α-AR are up-regulated in a variety of cardiomyopathies and may become more important than β-AR signal transduction in mediating catecholamine-induced increases in the inotropic state of the heart, particularly in ischemic heart disease. In this patient population, propofol may increase cardiac function without further altering Ca²⁺homeostasis, which could be beneficial to the patient.

As with all in vitro  methodologies and experimental approaches used to study cardiac function, there are some limitations.10,38The use of putative α-AR subtype–selective antagonists to focus on the signal transduction pathways of a particular receptor subtype prevents potential interactions among receptor subtypes. In addition, this in vitro  study only deals with intrinsic myocardial function, whereas changes in cardiac contractility in vivo  after propofol administration also depend on a variety of other factors, including venous return, afterload, and neurohumoral compensatory mechanisms. It is well established that species differences can contribute to the controversy surrounding specific cellular mechanisms regulating cardiac contractility. Also, there is no load on the isolated cardiomyocyte, which may be a limitation when comparing findings to studies using isometrically contracting cardiac muscle strips or Langendorff perfused hearts. Finally, the experimental conditions (temperature, stimulation frequency) used in this study do not parallel in vivo  conditions. However, the strength of this model is that we can directly assess the effects of α^1a-AR activation, propofol, and their interactions on cellular mechanisms that regulate cardiomyocyte contractile function.