Further investigations on the influence of protein phosphatases on the signaling of muscarinic receptors in the atria of mouse hearts

Abstract

The vagal regulation of cardiac function involves acetylcholine (ACh) receptor activation followed by negative chronotropic and negative as well as positive inotropic effects. The resulting signaling pathways may include G_i/o protein-coupled reduction in adenylyl cyclase (AC) activity, direct G_i/o protein-coupled activation of ACh-activated potassium current (I_KACh), inhibition of L-type calcium ion channels, and/or the activation of protein phosphatases. Here, we studied the role of the protein phosphatases 1 (PP1) and 2A (PP2A) for muscarinic receptor signaling in isolated atrial preparations of transgenic mice with cardiomyocyte-specific overexpression of either the catalytic subunit of PP2A (PP2A-TG) or the inhibitor-2 (I2) of PP1 (I2-TG) or in double transgenic mice overexpressing both PP2A and I2 (DT). In mouse left atrial preparations, carbachol (CCh), cumulatively applied (1 nM-10 µM), exerted at low concentrations a negative inotropic effect followed by a positive inotropic effect at higher concentrations. This biphasic effect was noted with CCh alone as well as when CCh was added after β-adrenergic pre-stimulation with isoprenaline (1 µM). Whereas the response to stimulation of β-adrenoceptors or adenosine receptors (used as controls) was changed in PP2A-TG, the response to CCh was unaffected in atrial preparations from all transgenic models studied here. Therefore, the present data tentatively indicate that neither PP2A nor PP1, but possibly other protein phosphatases, is involved in the muscarinic receptor-induced inotropic and chronotropic effects in the mouse heart.

Keywords: Heart; Inhibitor-2; Muscarinic receptor; PP1; PP2A; Protein phosphorylation; Transgenic mice.

Fig. 1

Summarized concentration–response curves for the effect of isoprenaline on force of contraction in electrically driven (1 Hz) left atrial preparations (A, B) and on the beating rate in right atrial preparations (C, D) from PP2A-TG, I2-TG, DT, and WT. Numbers in brackets give the number of experiments. Ctr is the pre-drug value. A Force of contraction in milli Newton (mN) and B shows the same data in % of control (Ctr) to visualize the efficiency of isoprenaline. *First p < 0.05 vs. Ctr; ^#p < 0.05 vs. WT. C Beating rate in beats per minute (bpm). *First p < 0.05 vs. Ctr; ^#p < 0.05 vs. WT and I2-TG. D The logarithm of the EC₅₀ values demonstrates the potency of isoprenaline to increase the beating rate in right atrial preparations. Significant differences (p < 0.05) are indicated by brackets

Fig. 2

Concentration–response curves for the effect of carbachol on force of contraction in electrically driven (1 Hz) left atrial preparations from PP2A-TG, I2-TG, DT, and WT. Numbers in brackets give the number of experiments. Ctr is the value before carbachol application. A Original recordings demonstrate the biphasic effect of carbachol. Ordinates: force of contraction (mN); horizontal bars: time (min). B Only the negative inotropic effect of carbachol is plotted and C only the positive inotropic effect of carbachol is plotted as delta milli Newton in % of pre-drug value, respectively, as described in the method section. *First p < 0.05 vs. Ctr; ^#p < 0.05 vs. WT and I2-TG

Fig. 3

Concentration–response curves for the effect of isoprenaline followed by carbachol on force of contraction in electrically driven (1 Hz) left atrial preparations from PP2A-TG, I2-TG, DT, and WT. Numbers in brackets give the number of experiments. Iso indicates the maximum response to isoprenaline (1 µM) and was used here as control for the carbachol application. A Original recordings demonstrate that the biphasic effect of carbachol is still present, but shifted to higher concentrations. Ordinates: force of contraction (mN); horizontal bars: time (min). B Only the negative inotropic effect of carbachol after pre-stimulation with Iso is plotted and C only the positive inotropic effect of carbachol after pre-stimulation with Iso is plotted as delta milli Newton in % of pre-drug value, respectively, as described in the method section. *First p < 0.05 vs. Iso; ^#p < 0.05 vs. WT and PP2A-TG

Fig. 4

Summarized concentration–response curves for the effect of carbachol alone (A) or after pre-stimulation with 1 µM isoprenaline (Iso) (B) on the beating rate in right atrial preparations from PP2A-TG, I2-TG, DT, and WT are shown. Numbers in brackets give the number of experiments. Ctr is the pre-drug value. Iso indicates the maximum response to isoprenaline and was used here as control for the carbachol application. *First p < 0.05 vs. Ctr or Iso

Fig. 5

Summarized concentration–response curves for the negative inotropic and chronotropic effect of the adenosine receptor agonist R-PIA in electrically driven (1 Hz) left atrial preparations (A, B) and in right atrial preparations (C), respectively, from PP2A-TG, I2-TG, and WT. Numbers in brackets give the number of experiments. Ctr is the value before R-PIA application. A Force of contraction in % of control (Ctr). B The logarithm of the EC₅₀ values demonstrates that the negative inotropic effect of R-PIA alone was not different between the mouse models. C Beating rate in beats per minute (bpm). *First p < 0.05 vs. Ctr

Fig. 6

Summarized concentration–response curves for the negative inotropic and chronotropic effect of the adenosine receptor agonist R-PIA after pre-stimulation with 1 µM isoprenaline (Iso) in electrically driven (1 Hz) left atrial preparations (A, B) and in right atrial preparations (C, D), respectively, from PP2A-TG, I2-TG, and WT. Numbers in brackets give the number of experiments. Iso indicates the maximum response to isoprenaline and was used here as control for the R-PIA application. A Force of contraction in % of Iso. B The logarithm of the EC₅₀ values demonstrates that the anti-adrenergic effect of R-PIA is enhanced in left atrial preparations from PP2A-TG compared to WT and I2-TG. Significant differences (p < 0.05) are indicated by brackets. C Beating rate in beats per minute (bpm). D The logarithm of the EC₅₀ values demonstrates that the negative chronotropic effect of R-PIA after pre-stimulation with Iso was not different in right atrial preparations. *First p < 0.05 vs. Iso; ^#p < 0.05 vs. PP2A-TG

Fig. 7

Protein phosphatase activity in whole heart homogenates. [³²P]-phosphorylase a was used as a substrate, mainly metabolized by PP2A and PP1. The release of [³²P]-Pi was measured as counts per microgram protein (cpm/µg protein). Of the protein phosphatase inhibitor okadaic acid, 10 nM was used to differentiate between PP1 and PP2A activity. Significant differences (p < 0.05) are indicated by brackets. N = 4–8