Introduction

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Stimulation of the parasympathetic postganglionic neurons causes the release of acetylcholine (ACh) that acts on muscarinic ACh receptors (mAChRs) on the myocardial cell membrane. There are five mammalian mAChR subtypes that preferentially couple to either the inhibition of adenylyl cyclase activity (M₂ and M₄ subtypes) or to the stimulation of phospholipase C (PLC) activity (M₁, M₃, and M₅ subtypes) (Bonner, 1989). The M₂ mAChR is the predominant muscarinic receptor subtype in mammalian heart (Brann et al., 1993; Caulfield, 1993). Activation of these M₂ mAChRs inhibits the activity of adenylyl cyclase, closes calcium channels, lowers the hyperpolarization-activated pacemaker current, and activates an inwardly rectifying potassium channel. These changes cause both negative chronotropic and inotropic effects on the heart (Caulfield, 1993).

Accumulating evidence indicates that other mAChR subtypes also contribute to the regulation of heart rate and contractility. The novel muscarinic agonist McN-A-343 has pressor activity in mammals (Roszkowski, 1961). When injected in dogs and cats, McN-A-343 raised blood pressure, an effect blocked by pretreatment with the muscarinic antagonist atropine. The McN-A-343-mediated hypertension in vivo was also antagonized by adrenergic blockade, suggesting that McN-A-343 is a muscarinic agonist that acts as a sympathomimetic agent. Pretreatment of dogs with reserpine blocks the pressor effect of McN-A-343. In awake rats, McN-A-343 induced hypertension and tachycardia, which are antagonized by propranolol (Martin, 1996). Adrenal demedullation had no effect on the tachycardia, whereas treatment with guanethidine suppressed both tachycardia and hypertension (Martin, 1996). Taken together, these results demonstrate that the muscarinic agonist McN-A-343 evokes both tachycardia and hypertension by releasing catecholamines from the sympathetic nerve endings, with only a minor role for the adrenal glands.

The muscarinic subtype that mediates the cardiostimulatory effect of McN-A-343 has not been clearly identified. The increase in blood pressure and heart rate after McN-A-343 injection in adult rats was blocked by low concentrations of the M₁/M₄ muscarinic antagonist pirenzepine (Hammer and Giachetti, 1982; Wilffert et al., 1983), suggesting that either the M₁ or M₄ subtype mediated the cardiostimulatory effects of McN-A-343. In addition, functional studies with cloned mAChR indicate that McN-A-343 can activate multiple muscarinic receptor subtypes with greatest efficacy at the M₄ receptor (Caulfield and Birdsall, 1998). Furthermore, the M₁, M₂, and M₄ mAChR subtypes are all expressed in sympathetic ganglia (Caulfield, 1993).

Pharmacological studies have also shown that muscarinic agonists can elicit a stimulatory effect on the mammalian heart. ACh caused a positive inotropic effect in human heart (Du et al., 1995) and in sheep and in canine cardiac Purkinje fibers (Gilmour and Zipes, 1985; Iacono and Vassalle, 1989). In isolated mouse atria ACh induces a biphasic effect on both maximum upstroke velocity of the action potential and inotropic response (Islam et al., 1998; Nishimaru et al., 2000), whereas in isolated perfused rat heart, ACh caused a dose-dependent increase in perfusion pressure (Hoover and Neely, 1997). It has been suggested that the positive inotropic action of ACh is a protective mechanism to prevent excessive inhibition of cardiac function at high concentrations of ACh (Pappano, 1991). However, the physiological effects of mAChR-mediated stimulation of the heart, particularly in vivo, are not well characterized and their mechanisms of action are poorly understood.

Other studies have demonstrated that other mAChR subtypes besides M₂ may be involved in cardiac function. For example, in canine atrial myocytes a novel K⁺ current is regulated by an M₃ mAChR subtype (Shi et al., 1999a,b). The ACh-mediated increase in rate of contraction in neonatal ventricular myocytes was insensitive to the M₂ antagonist AFDX-116 but antagonized by the M₁/M₄ antagonist pirenzepine (Gallo et al., 1993). In guinea pig cardiomyocytes carbachol mediated a pirenzepine-sensitive increase in the L-type Ca²⁺ channel activity (Gallo et al., 1993). These studies suggest that the M₁ or M₄ mAChR may be involved in the regulation of the heart. More recently, RT-PCR analysis has shown the expression of the M₁ mAChR subtype in rat and guinea pig ventricular cells (Gallo et al., 1993; Sharma et al., 1996) and the M₃ and M₄ mAChR subtypes in dog atrial cells (Shi et al., 1999b).

Although there is data indicating that the M₁ receptor may regulate cardiovascular function by acting either in the heart and/or the sympathetic ganglia, there appear to be considerable differences due either to species differences or lack of adequate selectivity of pharmacological agents. We took advantage of the M₁-KO mice previously generated in our laboratory (Hamilton et al., 1997) to determine the role of the M₁ mAChR in the regulation of cardiovascular function in mouse.

	Materials and Methods

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In Vivo Cardiodynamics. All procedures were approved by the University of Washington Institutional Animal Care and Use Committee. Mice (~6 months old) were anesthetized by intraperitoneal injection of tribromoethanol (Avertin, 0.4 mg/kg; Aldrich, Milwaukee, WI) and urethane--chloralose (750 mg/kg; 50 mg/kg). Mice were ventilated with a volume displacement ventilator (Harvard 687; Harvard Apparatus, Holliston, MA) and end respiratory CO₂ was recorded (96282-ND-PR; Columbus Instruments, Columbus, OH) and maintained between 2.8 and 3.2%. Body temperature was maintained at 37°C by using a rectal probe and proportional temperature controller (Harvard 7129; Harvard Apparatus) with a heating blanket. Where indicated, mice were bilaterally vagotomized to avoid vagal reflexes. The jugular vein was catheterized for drug injections (25 µl each).

RNA Extraction from Adult Heart. Total RNA from cortex, cerebellum, and whole heart from adult mice was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol with modification (Chomczynski and Sacchi, 1987).

RT-PCR. RT was performed at 42°C for 60 min by using 200 U of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) in a volume of 50 µl. The amplification reaction was carried out in the same tube in a final volume of 100 µl with 1.25 U of a thermostable Taq DNA polymerase (M186A; Promega). The temperature steps were 1 min at 94°C, 1 min at 56°C, and 1 min at 73°C. Forty-five cycles were performed and one additional cycle was added with a final elongation time of 10 min at 73°C. Each cardiac preparation was tested for DNA contamination in a parallel reaction performed in the absence of reverse transcriptase. The primers used for PCR were the 5'-primer 5'-ggatccggatccaaaggtggtggc-3' and the 3'-primer 5'-gaattcgaattctttcttggtgggcctcttgacgtg-3'. The reverse transcription was performed with the same 3'-primer.

Southern Blot. The RT-PCR products were separated on a 1.0% agarose gel by electrophoresis and transferred to a nylon membrane. A ³²P-labeled 1.8-kilobase KpnI-BamHI genomic fragment (10⁸ cpm/mg) containing the entire coding M₁ receptor region (Shapiro et al., 1988) was used as a probe.

[³H]QNB Binding Assay and Immunoprecipitation. Membrane isolation and solubilization, [³H]QNB binding assays, and immunoprecipitation with specific anti-M₁ and anti-M₂ antibodies were performed as described in Hamilton et al. (1997).

Phosphatidylinositol Hydrolysis Assay. WT and M₁-KO adult mice (~6 months old) were sacrificed by CO₂ intoxication. Hearts were quickly transferred to prewarmed Krebs' buffer containing 118 mM NaCl, 4.7 mM KCl, 3 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 0.5 mM EDTA, 25 mM NaHCO₃, and 10 mM glucose, pH 7.4 at 37°C. Atria and ventricles were cut into small pieces (1-3 mm) and incubated in 0.5 ml of oxygenated (95% O₂, 5% CO₂) Krebs' buffer containing 4 µCi/ml of [³H]myo-inositol (specific activity 16 Ci/mmol; Amersham Biosciences, Piscataway, NJ) for 90 min at 37°C with gentle agitation. Tissues were then washed with phosphate saline solution-LiCl solution (118 mM NaCl, 4.7 mM KCl, 3 mM CaCl₂, 1.2 mM KH₂PO₄, 10 mM glucose, 0.5 mM EDTA, 20 mM HEPES, and 50 mM LiCl) and incubated again for 30 min in this solution at 37°C. Carbachol (0.001-1 mM final concentration) was then added and the samples were incubated for an additional 30 min at 37°C.

Statistics. Data are expressed as mean ± S.E.M. Two-tailed unpaired t tests with 95% confidence limits or ANOVA was performed as indicated in the figure legends. The results were considered statistically significant if p < 0.05.

	Results

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M₁ mAChR Have a Catecholamine-Dependent Stimulatory Effect on Heart in Vivo

Basal Function. Cardiodynamic analyses were carried out to determine whether the lack of M₁ mAChRs affected basal cardiac function. The basal functions of the heart (LV dP/dt_max, DLVP, HR) were recorded in vagotomized mice before administration of drugs (Table 1). There is no statistical difference in these variables between the WT and M₁-KO mice, indicating that the lack of M₁ mAChRs did not alter basal cardiac function.

Vagotomy. To ensure that there were no differences in basal cardiac function between WT and mutant mice that were obscured by vagotomy, the change of heart rate due to vagotomy was also determined. These recordings were performed in separate groups of mice without cardiac catheterization. The heart rate was determined from ECG before and after bilateral vagotomy. Although a moderate tachycardia was observed as expected in both groups after vagotomy, the change was not statistically different between the WT (+69 ± 19 bpm; n = 4) and M₁-KO mice (+50 ± 11 bpm; n = 5).

McN-A-343 Induces Stimulatory Effects on Mouse Heart Mediated by -Adrenergic Receptors. Administration of the muscarinic agonist McN-A-343 (350 µg/kg) into bilateral vagotomized WT mice increased DLVP, LV dP/dt_max, and heart rate (Fig. 1, A-C). This effect was totally inhibited by pretreatment with the -adrenergic receptor antagonist propranolol (1 mg/kg).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Cardiac responses to McN-A-343 in bilateral vagotomized WT mice before and after -adrenergic receptor blockade with propranolol. Results are presented as the change (±S.E.M.) calculated from the baseline values of LV dP/dtmax (A), DLVP (B), and heart rate (C) preceding each drug injection (n = 6 WT mice). The muscarinic agonist McN-A-343 (350 µg/kg i.v.) induces both positive inotropic and chronotropic effects in WT mice (). In animals pretreated with 1 mg/kg i.v propranolol, administration of 350 µg/kg i.v. McN-A-343 no longer results in a significant change in hemodynamic variables. *, p < 0.05 McN-A-343 versus McN-A-343 + propranolol; ***, p < 0.001 McN-A-343 versus McN-A-343 + propranolol (t test). Administration of 1 mg/kg i.v. propranolol alone induced bradycardia. Effects of McN-A-343 in presence or in absence of propranolol were statistically different from those induced by propranolol alone. , p < 0.05 McN-A-343 versus propranolol alone; , p < 0.01 McN-A-343 versus propranolol alone.

Stimulatory Effect of McN-A-343 on Heart Is Mediated by M₁ mAChR. We took advantage of the M₁-KO mice previously generated in this laboratory to determine whether the M₁ muscarinic receptor is responsible for the cardiac stimulatory effects of McN-A-343. All mice were vagotomized bilaterally before drug injection to avoid vagal reflexes. A recording from a WT mouse treated with the agonist McN-A-343 is shown in Fig. 2 and a comparison of the responses from WT and M₁-KO mice is shown in Fig. 3, A-C. McN-A-343 caused a large increase in both DLVP and LV dP/dt_max as well as in the HR in the WT mice, with maximum increase observed at 350 µg/kg (27.5 ± 8.6 mm Hg in DLVP, 5.1 ± 0.78 m Hg/s in LV dP/dt_max, 71 ± 20 bpm in HR). Administration of McN-A-343 did not have a significant effect on any of the cardiodynamic variables in the M₁-KO mice.

View larger version (111K): [in this window] [in a new window]   Fig. 2.   Left ventricular pressure and LV dP/dt traces from bilateral vagotomized WT mouse before and after administration of McN-A-343. Administration of 350 µg/kg i.v. McN-A-343 resulted in a positive inotropic response.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A to C, cardiac responses to intravenous McN-A-343 in vagotomized WT and M1-KO mice. LV dP/dtmax (A), DLVP (B), and heart rate (C). WT () and M1-KO mice () were treated with increasing concentrations of McN-A-343 (0 to 350 and 0 to 1500 µg/kg, respectively). The results are expressed as the change from the basal values ± S.E.M. The results are statistically different between WT (n = 9) and M1-KO mice (n = 8). ****, p < 0.001 WT versus M1-KO; ***, p < 0.01 WT versus M1-KO; *, p < 0.05 WT versus M1-KO (ANOVA). The graphs show an McN-A-343-mediated increase in both inotropic and chronotropic responses in WT mice. The data demonstrate that the M1 mAChR-selective agonist McN-A-343 does not induce a positive regulation of the heart in M1-KO mice.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Hemodynamic responses to intravenous McN-A-343 in WT and M1-KO mice before and after bilateral vagotomy. MBP in WT mice (A), MBP in M1-KO mice (B), HR in WT mice (C), and HR in M1-KO mice (D). WT (A and C) and M1-KO mice (B and D) were treated with McN-A-343 (160 and 350 µg/kg) before ( for WT and  for M1-KO) and after ( for WT and  for M1-KO) bilateral vagotomy. The results are expressed as the increase from the basal value (before or after vagotomy) ± S.E.M. The results are statistically different (t test) between WT (n = 5) and M1-KO (n = 4). **, p < 0.01 WT versus M1-KO before vagotomy; *, p < 0.05 WT versus M1-KO before vagotomy; ##, p < 0.01 WT versus M1-KO after vagotmy; #, p < 0.05 WT versus M1-KO after vagotomy. The graphs show an McN-A-343-caused increase in blood pressure in WT mice; the M1-KO mice are not responsive to the M1 mAChR-selective agonist. The data also show that the McN-A-343-mediated effects are not the result of hypotension causing a baroreflex-induced tachycardia.

Nicotinic Responses Are Similar in Wild-Type and Knockout Mice. The agonist nicotine was injected at the end of each experiment as a positive internal control to test the effects of stimulation of nicotinic receptors in the sympathetic ganglia. As expected, nicotine induced a positive inotropic effect as well as tachycardia in both WT and M₁-KO mice (Fig. 5). The responses were not statistically different between the two groups. The effects of nicotine in M₁-KO mice demonstrate that the absence of a response to McN-A-343 was not due to an artifactual disruption of sympathetic effects on the heart.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Cardiac responses to intravenous nicotine in vagotomized WT and M1-KO mice. LV dP/dtmax (A), DLVP (B), and heart rate (C). Both WT (n = 8; ) and M1-KO mice (n = 7; ) were treated with increasing doses of nicotine (0.01-1 mg/kg). The results are expressed as the difference from basal values ± S.E.M. WT and M1-KO mice do not differ in their responses to nicotine (p > 0.05; ANOVA). This indicates that the lack of a response to the M1 receptor agonist McN-A-343 in WT mice was not due to an unresponsive preparation.

ACh-Induced Responses Are Similar in WT and M₁-KO Mice. To determine whether the lack of M₁ mAChR expression induced any change in M₂ mAChR responsiveness, ACh was injected in WT and M₁-KO mice. Endogenously released ACh acts on M₂ mAChR in the heart to induce both negative chronotropic and inotropic responses (Fleming et al., 1992; Caulfield, 1993). We therefore expected that administration of ACh would result in sinoatrial node bradycardia, which could be recorded on an ECG trace as a longer P-P wave interval and a moderate decrease in maximum LV dP/dt_max (the negative inotropic effect).

View larger version (84K): [in this window] [in a new window]   Fig. 6.   .Effect of ACh on atrioventricular conduction in WT (A) and M1-KO (B) mice. Mice were pretreated with 1 mg/kg propranolol before injections of ACh to avoid sympathetic reflexes. Before injection of ACh, P waves are observable before the QRS complex. Immediately after injection of 100 µg/kg i.v. ACh there are persistent P waves with irregular QRS complexes, indicating atrioventricular heart block. The atrial rate did not change after the injection of ACh, indicating the absence of a bradycardic effect on the sinoatrial node. The initial threshold effect of intravenous ACh is atrioventricular block without slowing of the atrial rate. Similar results were observed in mice that did not receive a propranolol pretreatment.

Mice Lacking M₁ mAChR Demonstrate No Change in M₂ mAChR Expression in Heart. M₁ mAChR Is Not Detectable in Wild-Type Mouse Heart

Immunoprecipitation. Because of the data indicating the presence of M₁ mAChR in the heart of rats and other mammals, we tested whether the M₁ receptor could be detected in WT mouse heart and whether the expression of mAChR was altered in M₁-KO mice. The total number of mAChR in atrial and ventricular tissues was determined using the nonselective muscarinic antagonist [³H]QNB. In both tissues, there was not a statistically significant difference in mAChRs number among the three genotypes: WT, heterozygote (HET), and M₁-KO (Fig. 7).

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Expression of muscarinic receptors subtypes in mouse heart. Total mAChR numbers were determined by [3H]QNB binding assay on tissue homogenates from WT (A), HET (B), and M1-KO (C) mouse hearts. In all cases values are the mean ± S.E.M. of four individual pairs of ventricles and three pools (5-6 animals/pool) of atrial tissues. [3H]QNB-labeled receptors from WT, HET, and M1-KO mouse heart were solubilized and immunoprecipitated with subtype-specific antisera. Values were compared by ANOVA. The total mAChR number is identical in all genotypes (p > 0.05). M2 mAChR number is also not different between the genotypes (p > 0.05). The amount of receptor precipitated by the anti-M1 mAChR antibody is near background levels and was not different between the three groups: p > 0.2 in atria (WT versus HET and WT versus M1-KO); p > 0.5 in ventricle (WT versus HET and WT versus M1-KO).

RT-PCR Experiments. RT-PCR was also used in an effort to detect low levels of M₁ mAChR mRNA in mouse heart. A representative result is shown in Fig. 8. As expected, a strong signal was present in the cortex where M₁ mAChR constitutes 50% of the total mAChR (Hamilton et al., 1997). No signal was detected in any tissue from the M₁-KO mouse, or in the cardiac tissues in the WT mouse.

View larger version (114K): [in this window] [in a new window]   Fig. 8.   RT-PCR and Southern blot analyses. Total RNA from WT and KO cortex (0.5 µg), and cerebellum, atria, and ventricles (1.5 µg each) were tested for M1 mAChR mRNA expression. Each RNA sample was reverse transcribed in the presence (+) or absence () of reverse transcriptase (to test for genomic DNA contamination) before amplification by primers designed in the third cytoplasmic loop. The PCR products were analyzed on a 1.0% agarose gel, transferred to nylon membrane, and hybridized with the 1.8-kilobase fragment encoding the entire coding region of the M1 mAChR. The M1 mAChR mRNA is expressed in both cortex and cerebellum but is not detectable in mouse heart.

Carbachol-Stimulated PLC Activity Is the Same in WT and M₁-KO Mice

Two mechanisms have been advanced to account for the stimulatory effects of muscarinic agonists on the heart: one involves an increase in intracellular Na⁺ concentration (by raising membrane permeability to Na⁺); the second mechanism involves stimulation of PLC, via Gq protein, which induces Ca²⁺ release from the endoplasmic reticulum via the inositol triphosphate released after PLC activation (Nathanson, 1987). M₁ mAChRs have been reported to mediate activation of PLC activity in guinea pig cardiomyocytes in culture (Gallo et al., 1993). We measured mAChR agonist-stimulated PLC activity in atrial and ventricular tissues from adult WT and M₁-KO mice (Fig. 9).

View larger version (21K): [in this window] [in a new window]   Fig. 9.   mAChR-mediated PLC activation in WT and M1-KO heart. The stimulation of PLC activity of ventricles (A) and atria (B) in response to muscarinic agonists was determined as described under Materials and Methods. The graphs show the percentage of increase in [3H]inositol phosphates formed after incubation with carbachol. The values are not statistically different (p > 0.05) between WT (filled) and M1-KO mice (open) both in ventricle (A) (n = 9 for WT and n = 8 for M1-KO mice; ANOVA) and in atria (B) (n = 7 in each group; t test). Results represent the average values ± S.E.M.

The cholinergic agonist carbachol (1 mM) induced an increase in PLC activity in both WT and M₁-KO cardiac tissues. In ventricle, increases of 128 ± 36 and 94 ± 27% over basal were observed in WT and M₁-KO, respectively (Fig. 9A). Incubation of atria with carbachol resulted in increases of 53 ± 12 and 43 ± 11% from WT and M₁-KO mice, respectively (Fig. 9B). If there were M₁ mAChRs in cardiac tissue, a greater PLC activity would be expected in WT than M₁-KO tissues; however, no significant difference in mAChR-mediated stimulation of PLC activity in hearts from WT and M₁-KO mice was observed.

	Discussion

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The main goal of this study was to determine the effects of elimination of the M₁ mAChRs on the function of the heart in vivo. Although the M₂ receptor is the main muscarinic subtype present in the heart and is responsible for the inhibitory effects of ACh, there have been conflicting data on the potential role of other subtypes in mediating effects of ACh on the heart.

We first compared the basal hemodynamic variables to determine whether the lack of M₁ mAChR had any influence on cardiac function at rest. Both developed left ventricular pressure and LV dP/dt_max as well as HR are similar between WT and M₁-KO mice, showing that the loss of M₁ mAChR has no effect on basal cardiac function. We then determined the effects of administration of the muscarinic agonist McN-A-343 in WT and M₁-KO mice. In WT mice, the agonist McN-A-343 was able to induce tachycardia, positive inotropism, and an increase in blood pressure. The increase in HR and MBP due to McN-A-343 administration observed in our study is in agreement with previous results reported on rat, dog, and cat (Roszkowski, 1961; Hammer and Giachetti, 1982; Wilffert et al., 1983).

None of the mice treated with McN-A-343 either before or after bilateral vagotomy showed a transient decrease in mean blood pressure before the McN-A-343-mediated increase in blood pressure. This indicates that McN-A-343 did not cause peripheral vasodilation and that the positive inotropic and chronotropic cardiac effects were not a baroreceptor reflex response to hypotension. On the other hand, the effect of McN-A-343 on the force of contraction in WT mice was blocked by the -adrenergic receptor antagonist propranolol. These observations together indicate that McN-A-343 probably acts at the postganglionic sympathetic nerves, which is consistent with previous work reported in other species (Roszkowski, 1961; Martin, 1996).

In pithed normotensive rats, the dose-dependent increase in blood pressure after the injection of McN-A-343 was strongly reduced in the presence of the M₁/M₄ antagonist pirenzepine (Hammer and Giachetti, 1982; Wilffert et al., 1983). It has also been previously shown that M₁-mediated suppression of the M-current potassium channel is completely absent in sympathetic ganglia from M₁-KO mice (Hamilton et al., 1997). However M₁, M₂, and M₄ mAChR are all expressed in the sympathetic ganglia (Caulfield, 1993). In the present study WT mice displayed a dose-dependent increase in both heart rate and LV dP/dt_max when injected with McN-A-343, but the M₁-KO mice did not. This clearly demonstrates that McN-A-343-induced responses are mediated by M₁ mAChR.

Although the apparent decreases in hemodynamic values observed at higher doses of McN-A-343 (500-1500 µg/kg) in M₁-KO mice might be due to a nonselective stimulation of cardiac M₂ mAChR, these decreases were not statistically different from the basal values.

Activation of nicotinic receptors in sympathetic ganglia induces catecholamine release from the endings of postganglionic cells, which results in an increase in both force and rate of contraction by stimulating the cardiac -adrenergic receptors. Administration of nicotine produced a similar response in both WT and M₁-KO mice, demonstrating that the lack of response to McN-A-343 in the KO mice was not due to a generalized defect in sympathetic transmission to the heart or an artifact of the surgical procedures.

Pharmacological studies have suggested a functional role for the M₁ mAChR in adult guinea pig (Gallo et al., 1993) and rat cardiomyocytes (Sharma et al., 1996). In rat cardiomyocytes, the stimulatory effects of mAChR agonists were significantly inhibited by antisense oligonucleotides directed against the M₁ mAChR subtype (Colecraft et al., 1998). In addition, RT-PCR analyses detected the expression of M₁ mAChR in adult rat and guinea pig ventricular myocytes in culture (Gallo et al., 1993; Sharma et al., 1996).

We performed biochemical and functional studies on mouse cardiac tissue to determine whether M₁ mAChRs were also expressed in the mouse heart. Immunoprecipitation analyses showed that the M₁-KO mice had unaltered levels of expression of M₂ receptors in the heart. Our results also show no statistically significant expression of the M₁ mAChR expression in WT mouse heart. We also used RT-PCR to detect M₁ mAChR mRNA in total RNA from WT heart. Despite multiple experimental conditions to increase sensitivity (two sets of primers to get a double amplification, radioactive PCR, variations in RT-PCR conditions, several RNA concentrations), we were not able to detect any signal in cardiac tissue from WT mice. Because we were able to detect M₁ mAChR mRNA in the cerebellum from WT mice, where the expression of M₁ mAChR represents less than 5% of total mAChR in cerebellum (Caulfield, 1993), the procedure was sensitive enough to detect very low levels of M₁ mAChR mRNA.

We also attempted to measure functional responsiveness of M₁ mAChRs in cardiac tissue. The mouse M₁ mAChR in transfected cells is preferentially coupled to the activation of PLC (Shapiro et al., 1988). Furthermore, M₁ mAChR is the main mAChR subtype coupled to activation of the PLC pathway in mouse cerebral cortical neurons (Hamilton and Nathanson, 2001). We measured the muscarinic agonist-mediated stimulation of PLC activity in heart from WT and M₁-KO mice. If M₁ mAChRs are present in cardiac WT tissue, PLC activity would be expected to be higher in WT tissue than in M₁-KO tissue. However, there was no significant difference in carbachol-mediated stimulation of PLC between WT mice compared with M₁-KO.

These biochemical results indicate that, in contrast to rat and guinea pig hearts, M₁ mAChRs are not expressed in the mouse heart. This is consistent with our physiological data showing that the effects of McN-A-343 are the consequence of activation of M₁ mAChR in the sympathetic ganglia (present study) and with the fact that McN-A-343 did not have any effect on the isolated mouse atria (Nishimaru et al., 2000). Our results also are in agreement with the report of Roszkowski (1961) who observed, in dog, an McN-A-343-induced rise in blood pressure in vivo but did not report any effect on isolated heart from dog, rabbit, and cat, suggesting the absence of M₁ mAChR in this tissue (Roszkowski, 1961; Shi et al., 1999b).

To determine whether the lack of M₁ mAChR in the KO mice affected the responsiveness of M₂ mAChR, ACh was injected in both WT and M₁-KO mice and the responsive effects were recorded by ECG. Both WT and M₁-KO mice responded to ACh with a similar threshold (data not shown), demonstrating that the lack of M₁ mAChR did not result in any change in M₂ mAChR-mediated response. However, the prominent effect of ACh on cardiovascular function in many mammals is to slow the rate of the sinus node depolarization. Unexpectedly the main and immediate effects of intravenous ACh on the mouse heart were the induction of atrioventricular conduction block, atrial flutter, or atrial fibrillation. Over a wide range of doses (0.01 pg/kg-200 µg/kg) sinus bradycardia was only barely and inconsistently detected. Similar Gi-coupled receptor-mediated arrhythmias have been also described in transgenic and knockout mice (Redfern et al., 2000; Kovoor et al., 2001). Because we never detected sympathetic reflex-mediated stimulation of the heart in mice treated with ACh in the absence of propranolol (data not shown), it is unlikely that the absence of ACh-induced bradycardia is the result of a sympathetic reflex that might have buffered the bradycardia. The absence of ACh-induced bradycardia could result from the high level of acetylcholinesterase in the sinus node (Loffelholz and Lindmar, 1994). This would prevent ACh from acting at the sinus node level, but would allow an action on the atrioventricular node where the concentration of acetylcholinesterase is lower (Loffelholz and Lindmar, 1994). This hypothesis is supported by the results of Walker et al. (1999) and Feniuk and Large (1975), who were able to detect bradycardia after injection of mice with the agonist methacholine (which is partially resistant to hydrolysis) or after injection of ACh into mice pretreated with the cholinesterase inhibitor physostigmine.

Because pharmacologically administered ACh did not appear to affect the sinoatrial node, the role of vagal tone in regulating murine heart rate was examined. WT and M₁-KO mice both exhibit tachycardia in response to bilateral vagotomy, indicating that the vagus did mediate a tonic inhibition of the sinoatrial node in resting mice.

In summary, we have shown that 1) McN-A-343 induces both tachycardia and a positive inotropic effect, as well as an increase in blood pressure, in vivo in mice, due to M₁ mAChR activation of postganglionic sympathetic neurons innervating the heart; 2) M₁ mAChRs are not detected in the WT mouse heart tissue; 3) the lack of M₁ mAChR does not modify M₂ mAChR cardiac responsiveness in vivo; and 4) pharmacological administration of ACh in mouse rarely results in sinus bradycardia but usually results in atrioventricular block, atrial fibrillation, or atrial flutter as the initial threshold effect.