Genetic disruption of G proteins, G(i2)alpha or G(o)alpha, does not abolish inotropic and chronotropic effects of stimulating muscarinic cholinoceptors in atrium

Abstract

Background and purpose: Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved.

Experimental approach: We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the alpha subunit of G(i2) protein (G(i2)alpha) or of the alpha subunit of G(o) protein (G(o)alpha). Preparations were stimulated with carbachol alone or after pretreatment with the beta-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca(2+)-channels in isolated ventricular cardiomyocytes were studied.

Key results: The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca(2+)-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied.

Conclusions and implications: These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G(i2)alpha and G(o)alpha mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.

Figure 4

Effect of isoprenaline in isolated right atria on rate of contraction (frequency) plotted in beats per minute (bpm). Ctr indicates control values (prior to drug addition). Littermate wild-type mice (+/+, open circles) are compared with G_i2α knockout (G_i2α−/−, A) or G_oα knockout mice (G_oα−/−, B). Numbers in brackets indicate number of mice studied. * denotes first significant difference versus pre-drug value (Ctr). No differences were detectable between wild type and knockout mice.

Figure 3

Effects of isoprenaline in isolated left atria on force of contraction (FOC) plotted in mN (B) or in percentage of maximum response to isoprenaline (A). Ctr indicates control values prior to drug addition. Littermate wild-type mice (+/+) are compared with G_i2α knockout (G_i2α−/−, A) or G_oα knockout mice (G_oα−/−, B). Numbers in brackets indicate number of mice studied. * denotes first significant difference versus pre-drug value. No differences were detectable between wild type and knockout mice.

Figure 2

Effects of carbachol alone in isolated right atria on rate of contraction (frequency) plotted in beats per minute (bpm). Ctr indicate control values (prior to drug addition). Littermate wild-type mice (+/+, open circles) are compared with G_i2α knockout (G_i2α−/−, A) or G_oα knockout mice (G_oα−/−, B). Numbers in brackets indicate number of mice studied. * denotes first significant difference versus pre-drug value. No differences were detectable between wild type and knockout mice.

Figure 1

Effects of carbachol alone in isolated left atria on force of contraction (FOC) plotted in mN (A) or in percentage of control values (Ctr, prior to drug addition, B). Littermate wild-type mice (+/+) are compared with G_i2α knockout (G_i2α−/−, A) or G_oα knockout mice (G_oα−/−, B). Numbers in brackets indicate number of mice studied. * denotes first significant difference versus pre-drug value. No differences were detectable between wild type and knockout mice.

Figure 5

Effect of isoprenaline alone (Iso, 30 nM) or in the additional presence of cumulatively applied carbachol in isolated right atria on rate of contraction (frequency) plotted in beats per minute (bpm). Ctr indicates control values (prior to drug addition). Littermate wild-type mice (+/+, open circles) are compared with G_i2α knockout (G_i2α−/−, A) or G_oα knockout mice (G_oα−/−, B). Numbers in brackets indicate number of mice studied. * denotes first significant difference of additionally applied carbachol versus Iso (alone) value. No differences were detectable between wild type and knockout mice.

Figure 6

Effects of carbachol alone or in the presence of isoprenaline (30 nM) in isolated left atria on force of contraction (FOC) plotted in mN (A) or in percentage of control values (Ctr, prior to drug addition, B). Solvent-treated G_i2α knockout (G_i2α−/−) mice are compared with PTX-treated mice (PTX). Numbers in brackets indicate number of mice studied.* denotes first significant difference versus pre-drug value (A) or versus Iso alone (B). + denotes significant differences versus solvent pretreatment.

Figure 8

Time-dependent effects of isoprenaline alone (Iso, 10 µM) and in the additional presence of carbachol (Carb) on current through the L-type Ca²⁺-channels (I_Ca, nA) in isolated, patch-clamped ventricular cardiomyocytes from littermate wild-type mice (G_o+/+, A) compared with G_oα knockout mice (G_oα−/−, B). Representative tracings from five cardiomyocytes from three different G_oα+/+ and G_oα−/− hearts are shown.

Figure 7

Time-dependent effects of isoprenaline alone (Iso, 10 µM) and in the additional presence of carbachol (Carb) on current through the L-type Ca²⁺-channels (I_Ca, nA) in isolated, patch-clamped, ventricular cardiomyocytes from littermate wild-type mice (G_i2α+/+, A) compared with G_i2α knockout mice (G_i2α−/−, B). Representative tracings from five cardiomyocytes from three different G_i2α+/+ and G_i2α−/− hearts are shown.