Biphasic redistribution of muscarinic receptor and the altered receptor phosphorylation and gene transcription are underlying mechanisms in the rat heart during sepsis

Abstract

Objective: The purpose of this study was to investigate intracellular redistribution of muscarinic cholinergic receptor (m2AChR) and the roles of receptor phosphorylation and gene transcription as underlying mechanisms in the rat heart during different phases of sepsis. Methods: Sepsis was induced by cecal ligation and puncture (CLP). The density of m2AChR in the sarcolemmal and light vesicle fractions was studied using [³H]-quinuclidinyl benzilate ([³H]-QNB). Phosphorylation of m2AChR was studied by labeling of the myocardial ATP pool by perfusing isolated hearts with [³²P]H₃PO₄ followed by identification of the phosphorylated m2AChR with SDS–PAGE. The steady-state level of m2AChR mRNA was determined by RT-PCR and Southern blot analysis. Results: Septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP, early sepsis) and a subsequent hypocardiodynamic (18 h after CLP, late sepsis) state. During early sepsis, the B_max for [³H]-QNB binding was increased in sarcolemma (+69%) but decreased in light vesicles (−22%), whereas during late sepsis, the B_max was decreased in sarcolemma (−20%) but increased in light vesicles (+32%). The sum of B_max for sarcolemmal and light vesicle fractions was increased during early sepsis (+43%) but decreased during late sepsis (−14%). The phosphorylation of m2AChR was decreased during early sepsis (−73%) but increased during late sepsis (+36% to +90%). The m2AChR mRNA abundance was increased during early sepsis (+52%) but decreased during late sepsis (−28%). Conclusions: The m2AChR in the rat heart was externalized from light vesicles to sarcolemma (overexpression) during early sepsis but internalized from surface membranes to intracellular sites (underexpression) during late sepsis. Furthermore, changes in the receptor phosphorylation and gene transcription are responsible for the biphasic redistribution and the altered expression of m2AChR in the rat heart during the progression of sepsis.

Gene expression, Muscarinic (ant)agonists, Protein phosphorylation, Receptors, Sarcolemma, Septic shock

Time for primary review 22 days.

1 Introduction

Cardiac functions are co-regulated by sympathetic–adrenergic and parasympathetic–acetylcholinergic systems [1]. The major effects of parasympathetic vagal stimulation on the heart include bradycardia and negative inotropic action. The inhibitory effects of cholinergic stimulation on the ventricles and His–Purkinje system are much more prominent following β-adrenergic stimulation [1,2]. Recent studies have indicated that activation of muscarinic acetylcholine receptor (mAChR) in the heart triggers a variety of signal transduction pathways, resulting in either positively or negatively inotropic/chronotropic effects [1–3]. mAChR consists of five different genes, m1, m2, m3, m4 and m5, with m1 and m2 subtypes being expressed in the mammalian heart. Following agonist stimulation, the m2 subtype (m2AChR) exerts an inhibitory response while the m1 subtype (m1AChR) elicits an excitatory effect. Furthermore, they undergo translocation into a specific subcompartment from the surface membrane, a characteristic of many G protein-coupled receptors [2,4–6].

Clinically, sepsis is a two-phase process in which patients initially go through a hyperdynamic phase and subsequently a hypodynamic phase [7]. Recent studies from this laboratory on the pathogenesis of cardiac dysfunction during sepsis have disclosed that β-adrenergic receptor (βAR) and α-adrenergic receptor (αAR) in the heart were externalized from light vesicles to sarcolemma during the early hyperdynamic phase of sepsis while they were internalized from surface membranes to intracellular sites during the late hypodynamic phase of sepsis [8,9]. Further investigation reveals that altered phosphorylation of receptor proteins is a mechanism responsible for the biphasic redistribution of βAR in the rat heart during different phases of sepsis [10]. Since m2AChR, βAR and αAR possess a common coupling system including the GTP binding protein, and are regulated by similar phosphorylation reactions [11–13], it is conceivable that cardiac m2AChR may undergo a biphasic intracellular redistribution as of βAR and αAR during the two distinct cardiodynamic phases of sepsis. Accordingly, the current study dealing with intracellular redistribution of m2AChR and its underlying mechanisms was undertaken in an attempt to understand the pathophysiology of myocardial dysfunction during the progression of sepsis.

2 Methods

2.1 Animal model

All animal experiments in this study were performed with the approval of the animal care committee of Saint Louis University School of Medicine, and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague–Dawley rats weighing from 270 to 320 g were used. All animals were fasted overnight with free access to water. They were divided into three groups: control, early sepsis, and late sepsis. Sepsis was induced by cecal ligation and puncture (CLP) as described by Wichterman et al. [14] with minor modification. Under halothane anesthesia, a laparotomy was performed and the cecum was ligated with a 3-0 silk ligature and punctured twice with an 18-gauge needle. The cecum was then returned to the peritoneal cavity and the abdomen was closed in two layers. Control rats were sham-operated (a laparotomy was performed and the cecum was manipulated but neither ligated nor punctured). All animals were resuscitated with 4 ml per 100 g body weight of normal saline at the completion of surgery and also at 7 h postsurgery. Animals were fasted but had free access to water after operative procedures. Hearts were removed from control and septic animals 9 or 18 h after the operation under chloralose and urethane anesthesia and were then subjected to perfusion as described in Section 2.2. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The mortality rates were 0% for control (none of the 66 animals died within 18 h after sham operation), 14% for early sepsis (11 out of 76 animals died within 9 h after CLP), and 29% for late sepsis (27 out of 93 animals died within 18 h after CLP). The septic rat hearts depict two dynamic states: an initial hypercardiodynamic (9 h after CLP) followed by a hypocardiodynamic state (18 h after CLP). The hypercardiodynamic state was characterized by elevated cardiac output, heart rate, and +LV dP/dt_max during the early phase of sepsis while the hypocardiodynamic state was characterized by diminished cardiac output, heart rate, mean arterial blood pressure, +LV dP/dt_max and –LV dP/dt_max during the late phase of sepsis.

2.2 Isolated heart perfusion

Isolated heart perfusion was performed as described previously [10]. Hearts removed from control or septic rats were retrogradely perfused by the Langendorff technique using Krebs–Henseleit (KH) buffer for 5 min (first perfusion), followed by a 25-min perfusion with KH buffer in the presence of 0.1 μM prazosin and 1 μM propranolol (second perfusion). At the end of the second perfusion, the hearts were perfused for 5 min with KH buffer (third perfusion). In some experiments, 1 μM acetylcholine or 0.1 μM atropine was present during the third perfusion. At the end of the third perfusion, cardiac ventricles were rapidly frozen with liquid nitrogen and then pulverized. The pulverized myocardium was then used for the preparation of sarcolemmal and light vesicle fractions, and subsequently, for the m2AChR binding assay.

2.3 Muscarinic acetylcholine receptor binding assay

m2AChR binding assays in the sarcolemmal and light vesicle fractions were carried out using [³H]-quinuclidinyl benzilate ([³H]-QNB) as a radioligand according to a procedure described by Fields et al. [15] with modification. The sarcolemmal and light vesicle fractions were prepared as described previously [8–10]. The assay mixture (0.25 ml) for m2AChR binding contained 10 mM MgCl₂, 50 mM NaH₂PO₄/Na₄P₂O₇, (pH 7.4), 0.025–3.0 nM [³H]-QNB (43 Ci/mmol), and either in the absence or presence of 5 μM of unlabeled himbacine (a m2-subtype selective antagonist [16,17]. The binding assay was initiated by the addition of sarcolemmal membranes (40 μg protein) or light vesicles (60 μg protein) and proceeded for 40 min at 37°C. At the end of each incubation, the reaction mixture was diluted with 4 ml of ice-cold washing buffer (10 mM MgCl₂, 50 mM NaH₂PO₄/Na₄P₂O₇, pH 7.4) and filtered through a 0.45-μm glass fiber filter paper (Baxter Healthcare) under suction. The filter paper was washed, dried, and the radioactivity was then determined. The specific binding was defined as the bound radioactivity displaceable by 5 μM of unlabeled himbacine.

2.4 Phosphorylation of muscarinic acetylcholine receptor

Hearts were perfused as described earlier in Section 2.2 except that [³²P]H₃PO₄ was present during the second perfusion in order to label the tissue ATP pool. Following the perfusion, the sarcolemmal and light vesicle fractions were prepared as described previously [8–10]. The ATP content of the myocardium was quantified by the method of Adams [18]. The specific radioactivity of the γ-phosphate group of ³²P-labelled ATP was determined by the method of Hawkins et al. [19]. The phosphorylated m2AChR were separated and analyzed by SDS–PAGE (7.5–9.5% acrylamide gradient gel) and autoradiographied as described previously [8–10]. The covalent incorporation of [γ-³²P]ATP into m2AChR was quantified by counting the radioactivity of the dried gel tracks cut into 3-mm slices [8,10].

2.5 Determination of the steady-state level of m2AChR mRNA by RT-PCR and Southern blot analysis

The steady-state level of m2AChR mRNA was determined by RT-PCR based on the method of Hardouin et al. [20] using total cellular RNA extracted from the control and septic rat hearts. The sense primer (5′-GAA CAC AAC AAG ATC CAG AAT GGC AAG-3′) and the antisense primer (5′-CGG AGC ATG GGC GCA ATG ATA G-3′) were designed based on the published cDNA sequences [21] and synthesized by Bio-Synthesis (Lewisville, TX). Cycling conditions consisted of a single 5-min heating step at 95°C, followed by 35 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Initially, the number of cycles needed for exponential amplification was titrated. The PCR-amplified product was then used for the Southern blot analysis. For internal standard, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was simultaneously amplified using 5′-TGA AGG TCG GAG TCA ACG GAT TTG GT-3′ as a sense primer and 5′-CAT GTG GGC CAT GAG GTC CAC CAC-3′ as an antisense primer.

Southern blot analysis was performed by the method of Sambrook et al. [22]. The probe specific for m2AChR was a 660 bp 3′-terminal StyI–PstI fragment of the rat m2AChR cDNA purchased from American Type Culture Collection (Manassas, VA) and was labeled with enhanced chemiluminescent (ECL) random-prime labeling and detection systems (Amersham Life Science). The probe specific for G3PDH (5′-CAC GGA AGG CCA TGC CAG TGA GCT TCC CGT-3′) was labeled with ECL 3′-oligolabelling and detection systems (Amersham Life Science). A 1:1000 dilution of an antifluorescein antibody conjugated to horseradish peroxidase (Amersham Life Science) was used. Autoradiographs were scanned and the relative densities were quantified as described earlier [10]. The mRNA levels were normalized relative to the level of G3PDH to correct for the potential difference in the amount of RNA used and the DNA amplified.

2.6 Other measurements and statistical analysis

(Na⁺+K⁺)-ATPase activity, Ca²⁺-ATPase activity, and protein content of the myocardium were determined as described previously [8–10]. The statistical analysis of the data was performed using one-way ANOVA followed by Student–Newman–Keuls tests. A P value of less than 0.05 was considered statistically significant.

2.7 Materials

Quinuclidinyl benzilate, l-[benzilic-4,4′-³H]-, (43.0 Ci/mmol) was purchased from DuPont New England Nuclear. Acetylcholine, (+)-himbacine, atropine, prazosin, propranolol, and R-(−)-3-quinuclidinyl benzilate were products of Sigma Chemical. Other chemicals and reagents were of analytical grade.

3 Results

Figs. 1 and 2 show the effects of sepsis on the dynamics of m2AChR in the sarcolemmal and light vesicle fractions prepared from non-perfused hearts based on [³H]-QNB binding studies. [³H]-QNB bindings to cardiac membranes exhibit a saturable process with a single-component binding characteristic for all three experimental groups (Figs. 1A and 2A). In the sarcolemmal membrane fraction (Fig. 1), the maximum binding capacity (B_max) calculated from Scatchard plot (Fig. 1B) was increased by 60% (P<0.01) during early sepsis but decreased by 28% (P<0.01) during late sepsis (576±15, 922±26, and 414±11 fmol/mg for control, early sepsis, and late sepsis, respectively). The affinity [the reciprocal of the dissociation constant (K_d)] for [³H]-QNB binding in sarcolemmal membranes remained unaffected during early and late phases of sepsis. In the light vesicle fraction (Fig. 2), the B_max for [³H]-QNB binding (Fig. 2B) was decreased by 22% (P<0.01) during early sepsis but increased by 39% (P<0.01) during late sepsis (152±5, 119±6, and 212±9 fmol/mg for control, early sepsis, and late sepsis, respectively). The affinity for [³H]-QNB binding in light vesicles was unaltered during early and late sepsis. These data indicate that m2AChR in the rat heart was externalized from light vesicle to sarcolemmal membrane during early sepsis but internalized from surface membrane to intracellular site during late sepsis. The sum of B_max of sarcolemmal membrane and light vesicle fractions was increased by 43% (P<0.01) during early sepsis but decreased by 14% (P<0.01) during late sepsis (728±13, 1041±29, and 627±18 f mol/mg for control, early sepsis, and late sepsis, respectively). These data seem to imply that, in addition to the intracellular redistribution, m2AChR was overexpressed during the early phase whereas it was underexpressed during the late phase of sepsis.

Fig. 2

Effects of sepsis on [³H]-QNB binding in light vesicles isolated from non-perfused hearts. [³H]-QNB binding was performed as described in Methods. (A) Vertical bars indicate standard errors of the mean (n=6). (B) Scatchard plots using data obtained from A. For other symbols, see the legend to Fig. 1. * P<0.05, * P<0.01 as compared with the control group.

Fig. 1

Effects of sepsis on [³H] -QNB binding in sarcolemmal membranes isolated from non-perfused hearts. [³H] -QNB binding was performed as described in Methods. (A) Early sepsis (ES) and late sepsis (LS) refer to measurements performed at 9 h and 18 h, respectively, after cecal ligation and puncture. Vertical bars indicate standard errors of the mean (n=6). (B) Scatchard plots using data obtained from A. * P<0.05, * P<0.01 as compared with the control group.

It should be pointed out that when heart was perfused in the absence of acetylcholine, the B_max for [³H]-QNB binding was increased by 69% (P<0.01) during early sepsis but decreased by 20% (P<0.05) during late sepsis in the sarcolemmal fraction. In the light vesicle fraction, however, the B_max for [³H]-QNB binding was decreased by 22% (P<0.05) during early sepsis but increased by 32% (P<0.01) during late sepsis. The K_d values were unaffected during the early and the late phases of sepsis in both membrane fractions. These data demonstrate that changes in the dynamics of m2AChR during the progression of sepsis in perfused heart experiments were almost identical to those observed in the non-perfused hearts (Figs. 1 and 2). When the heart was perfused in the presence of acetylcholine (1 μM), the B_max for [³H]-QNB binding was significantly reduced in the sarcolemmal fraction as compared to that in the absence of acetylcholine for each experimental group: i.e., 45% decrease (P<0.01) in control; 51% decrease (P<0.01) in early sepsis, and 28% decrease in late sepsis (P<0.05). In the light vesicle fraction, however, the B_max was significantly increased by acetylcholine in the control and early septic experiments (33% increase in control, 86% increased in early sepsis) with no change in the B_max for late septic experiment. The lack of stimulatory effect of acetylcholine on the internalization of m2AChR during late sepsis indicates that m2AChR in late stage of sepsis was maximally internalized. It is of interest to note that during late sepsis, perfusion of atropine (0.1 μM) reversed the sepsis-induced internalization, suggesting that sepsis-induced internalization of m2AChR during the late stage of sepsis may be mediated via the same mechanism as of agonist-induced internalization of m2AChR.

Figs. 3 and 4 depict changes in the phosphorylation of m2AChR in the sarcolemmal (Fig. 3) and light vesicle (Fig. 4) fractions during the early and the late phases of sepsis. In the sarcolemmal fraction (Fig. 3), the incorporation of [γ-³²P]ATP into 80 kDa peptides was decreased by 73% (P<0.01) during early sepsis but increased by 93% (P<0.01) during late sepsis. In the light vesicle fraction (Fig. 4), the incorporation of [γ-³²]ATP into 80-kDa peptides was decreased by 73% (P<0.01) during early sepsis but increased by 36% (P<0.01) during late state of sepsis. The phosphorylation of m2AChR in the sarcolemmal and light vesicle fractions was stimulated by acetylcholine (1 μM) within each experimental group. In the sarcolemmal fraction (Fig. 3), there were 175, 820, and 77% increases, respectively, in the control, the early septic, and the late septic experiments, upon acetylcholine stimulation (Fig. 3B). In the light vesicle (Fig. 4), there were 76, 557, and 82% increases, respectively, in the control, the early septic, and the late septic experiments, upon acetylcholine stimulation (Fig. 4B). The acetylcholine-induced increases in the phosphorylation of m2AChR in the sarcolemmal and light vesicle fractions during the late stage of sepsis were abolished by atropine (0.1 μM) (Figs. 3A and 4A). These data indicate that the phosphorylation of m2AChR in cardiac sarcolemma and light vesicles was decreased during early sepsis while it was increased during late sepsis. Furthermore, the sepsis-induced increase in the phosphorylation of m2AChR during late stage of sepsis may be mediated through a mechanism similar to the agonist-induced phosphorylation of m2AChR.

Fig. 4

Changes in the phosphorylation of m2AChR in the light vesicle fraction of the rat heart during different phases of sepsis. Fig. 4A depicts a representative autoradiograph and Fig. 4B summarizes quantitative results obtained from SDS–PAGE analysis (n=6). The experiments were carried out as described under Methods. For symbols and abbreviations, see legend to Fig. 3.

Fig. 3

Changes in the phosphorylation of m2AChR in the sarcolemmal membrane of the rat heart during different phases of sepsis. The experiments were carried out as described under Methods. Fig. 3A depicts a representative autoradiograph and Fig. 3B summarizes quantitative results obtained from SDS–PAGE analysis (n=6). (+) Signifies that hearts were perfused in the presence, while (–) signifies in the absence of acetylcholine (ACh) (1 μM) or atropine (0.1 μM) during the third perfusion period. Numbers on the left represent the position of mass standards. m2AChR=muscarinic cholinergic receptor; ES=early sepsis; LS=late sepsis. * P<0.01.

Fig. 5 illustrates the relationship between changes in the intracellular redistribution of m2AChR and alterations in the receptor phosphorylation in the rat heart during different phases of sepsis. In the sarcolemmal fraction, the increase in the number of m2AChR (externalization) during early sepsis coupled with a decrease in the receptor phosphorylation, while the decrease in the number of m2AChR (internalization) during late sepsis accompanied with an increase in the receptor phosphorylation (Fig. 5A). In the light vesicle fraction, the decrease in the number of m2AChR (externalization) during early sepsis coincided with a decrease in the receptor phosphorylation, while the increase in the number of m2AChR (internalization) during late sepsis correlated with an increase in the receptor phosphorylation (Fig. 5B). Since receptor phosphorylation–dephosphorylation has been considered to be a common mechanism through which recycling of receptors can be regulated [12,13,23,24], our data presented in Fig. 5 strongly suggest that a decrease in the receptor phosphorylation is a mechanism for the externalization of m2AChR during early sepsis while an increase in the receptor phosphorylation is a mechanism for the internalization of m2AChR during late sepsis.

Fig. 5

The relationship between changes in the intracellular redistribution of m2AChR and alterations in the receptor phosphorylation in rat hearts during different phases of sepsis. The figures were constructed using data presented in Figs. 3 and 4 and those presented in the third paragraph of the Results section in which hearts were perfused in the absence of acetylcholine during the third perfusion period. CLP=cecal ligation and puncture. Fig. 5A represents sarcolemma while Fig. 5B represents light vesicle. Vertical bars indicate standard errors of the mean (n=6). * P<0.05; *P<0.01 as compared to zero time in each specific group.

Fig. 6 depicts the amplified PCR products derived from specific m2AChR primers in the control rat heart. No amplified DNA products could be revealed after RNase A digestion (lane 1; Fig. 6A). This confirms the absence of the contamination of DNA in the RNA samples. The amplified DNA products were increased proportionally with the increasing concentrations of template RNA (lanes 2, 3, 4, 5, 6 contained 100, 200, 400, 600, 800 ng of RNA, respectively; Fig. 6A). The amplified PCR products were increased proportionally from 20 to 38 cycles (Figs. 6B and 6C). These results indicate that the RT-PCR used in this study is sufficiently sensitive and accurate to detect changes in the m2AChR mRNA level under our experimental conditions. Based on these results, a condition consisting of 35 cycles for m2AChR mRNA and 26 cycles for G3PDH mRNA was adopted for comparison of changes in the steady-state levels of m2AChR mRNA among the control, the early septic, and the late septic experiments.

Fig. 6

(A) A representative picture of ethidium bromide stain of agarose gels for the assessment of DNA-free RNA preparations from control rat hearts. (+) Indicates in the presence while (–) represents in the absence of RNase A (1 U). (B) Ethidium bromide stain of an representative agarose gel after different cycles of amplification. (C) Laser densitometric analysis of the cycle-dependent amplification of DNA product using data obtained from Fig. 6B. RT-PCR was performed using 100 ng RNA and 20–38 amplification cycles. After each cycle, 15-μl aliquots were taken from the reaction mixture and were separated on 2% agarose gels with 0.1% of ethidium bromide. The gel was laser scanned and the relative densities of DNA product were quantified by a Jandel Scientific Software program. m2AChR, muscarinic cholinergic receptor.

Fig. 7 shows RT-PCR and Southern blot analysis of the steady-state levels of m2AChR mRNA in the control and septic rat hearts. Using m2AChR-specific primers, the reverse transcription and amplification of total RNA extracted from all experimental groups resulted in a single band of the expected size of 790 bp (Fig. 7A). Analyses of the densitometric signals reveal that the steady-state level of m2AChR mRNA was increased by 52% (P<0.01) during the early but decreased by 28% (P<0.01) during the late phases of sepsis (Fig. 7B). It should be mentioned that the yields and the purities of total RNA remained unaltered among control, early septic, and late septic groups, indicating that changes in the steady-state level of m2AChR mRNA were not a result of alterations in the isolation procedure for cardiac RNA. These data demonstrate that the m2AChR gene transcript in the rat heart was overexpressed during the early phase but underexpressed during the late phase of sepsis. These findings indicate that the altered receptor gene transcription is an additional mechanism regulating m2AChR expression during septic shock. The findings thus provide an explanation as to why the combined receptor density of the two subcellular fractions was increased during the early, but decreased during the late phases of sepsis.

Fig. 7

(A) A representative autoradiograph of RT-PCR and Southern blot analysis. (B) Changes in the steady-state levels of m2AChR mRNA in the rat heart during sepsis. RT-PCR and Southern blot analysis were performed as described in the Methods. All m2AChR mRNA levels were normalized relative to the level of G3PDH to correct for potential differences in the amount of RNA used and the DNA amplified. (C) Control; ES, early sepsis; LS, late sepsis.

4 Discussion

In this study, identical approaches including septic rat model, cardiac membrane preparation, and isolated heart perfusion technique employed for the studies of βAR and αAR [8–10], were adopted for the studies of m2AChR. It should be mentioned that the receptor binding assay was conducted using unlabeled himbacine, a m2-subtype selective antagonist, to displace [³H]-QNB binding, indicating that the m2AChR we studied represents m2AChR. The results demonstrate that the m2AChR underwent a biphasic intracellular redistribution in rat heart during the progression of sepsis, that is, an externalization from light vesicle to sarcolemmal membrane during the early hyperdynamic phase followed by an internalization from surface membrane to intracellular site during the late hypodynamic phase of sepsis. Furthermore, the externalization of m2AChR during the early phase of sepsis was coupled with a decrease in receptor phosphorylation while the internalization of m2AChR during the late phase of sepsis coincided with an increase in m2AChR phosphorylation.

The functional sequelae of the biphasic redistribution of m2AChR in the heart in relation to the alterations of cardiodynamic state during the progression of sepsis appear to be quite complex. The m2AChR mediates parasympathetic control of cardiac inotropy and chronotropy [1,4]. The primary effects of m2AChR stimulation are opposite to those of βAR stimulation. Under the stimulatory influence of βAR, activation of m2AChR results in negative inotropic and chronotropic effects on the heart through activation of Gαi and inhibition of adenylate cyclase [1,4]. However, activation of m2AChR also couples to the activation of phosphoinositide turnover and opening of the Ca²⁺ channel and the Na⁺ channel, which appears to be positively inotropic [1,2,25]. It should be mentioned that when extending the dual negative and positive inotropic effects of m2AChR mediation from animal studies to human biology, caution should be made since such an action on myocardial function has not been demonstrated without doubt in humans. Under pathological conditions, the relative contribution between the antagonistic interaction between m2AChR and βAR stimulation to the control of cardiac function is still a matter of debate [26,27]. Parasympathetic regulation of cardiac function via m2AChR mediation has been reported to be co-compromised with βAR during various disease states. Parallel increases in the densities of m2AChR and βAR was observed in acute myocardial ischemia [28], while parallel decreases in the densities of m2AChR and βAR was seen in cardiac hypertrophy [29]. The fact that the externalization of m2AChR during early hyperdynamic phase of sepsis (as observed in this study) occurred concomitantly with the externalization of βAR [8] while the internalization of m2AChR during late hypodynamic phase of sepsis (current study) took place simultaneously with internalization of βAR [8], suggests that the intracellular redistribution of m2AChR may provide an alternative means of establishing a new equilibrium for the adrenergic system.

The mechanism responsible for the biphasic translocation of cardiac m2AChR between the surface membrane and the intracellular location during the progression of sepsis is not clearly understood. Studies on the regulation of receptor function have indicated that increased phosphorylation of m2AChR leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered compartment, and once the receptors are sequestered, the phosphorylation is reversed, perhaps enabling the receptors to translocate back to the cell surface [12,13,23,24]. In the current study, we found that the incorporation of [γ-³²P]ATP into m2AChR in sarcolemma and light vesicle fractions was decreased by 73% during early hyperdynamic phase of sepsis while it was increased by 36–93% during late hypodynamic phase of sepsis. Furthermore, the decrease in m2AChR phosphorylation correlated with its externalization from light vesicle to sarcolemma while the increase in m2AChR phosphorylation coupled with its internalization from surface membrane to intracellular site. These data demonstrate that the altered phosphorylation/dephosphorylation of receptor protein is a mechanism responsible for the biphasic expression of m2AChR in the heart during the two distinct cardiodynamic phases of sepsis. Our findings, however, do not provide information in regard to whether the altered phosphorylation of m2AChR during the progression of sepsis is involved with alteration in m2AChR kinase and/or arrestin binding [6,13,23].

In addition to the post-translational regulation, i.e., divalent modification of receptor protein by phosphorylation/dephosphorylation, the altered m2AChR density can be regulated at the transcriptional level. In human embryonic lung fibroblasts, m2AChR protein and mRNA levels were down-regulated by multifunctional cytokines such as TGF-β, TNF-α and interleukin 1β through a reduction in m2AChR gene transcription [30]. In cardiac hypertrophy, the reduction in the m2AChR density was accompanied by a decrease in m2AChR mRNA level [29]. In septic shock, as reported in this study, the steady-state level of m2AChR mRNA was increased by 52% during the early phase while it was decreased by 28% during the late phase of sepsis. Our findings thus provide direct evidence of transcriptional regulation for cardiac m2AChR during the progression of sepsis.

In summary, the present findings illustrate that changes in the expression of m2AChR in the rat heart during the two distinct cardiodynamic phases of sepsis occur concomitantly with changes in the receptor subset expression on βAR (8), αAR (9), and (Na⁺+K⁺)-ATPase [31]. Although the integration of the findings of the concordant changes in the receptor subset expression on βAR, αAR, m2AChR, and (Na⁺+K⁺)-ATPase in regard to what it means to human pathophysiology requires further clarification, one may speculate that during the early hypercardiodynamic phase of sepsis, the increase in the expression of (Na⁺+K⁺)-ATPase and that of m2AChR may counterbalance the overexpression of βAR and αAR, or alternately, may represent an adaptive change in response to the overexpression of βAR and αAR. While during the late hypocardiodynamic phase of sepsis, the underexpression of the receptor subsets on βAR, αAR, m2AChR, and (Na⁺+K⁺)-ATPase may reflect the failing status of the myocardium because the effectiveness of its controlling mechanisms has been compromised.

Acknowledgements

This work was supported by HL-30080 from the National Heart, Lung, and Blood Institute, and GM-31664 from the National Institute of General Medical Sciences, National Institutes of Health.