Abstract
Muscarinic receptors (MR) are main cardioinhibitory receptors. We investigated the changes in gene expression, receptor number, echocardiography, muscarinic/adrenergic agonist/antagonist changes in heart rate (HR) and HR biorhythm in M2 KO mice (mice lacking the main cardioinhibitory receptors) in the left ventricle (LV) and right ventricle (RV). We hypothesize that the disruption of M2 MR, key players in parasympathetic bradycardia, would change the number of receptors with antagonistic effects on the heart (β1- and β2-adrenoceptors, BAR), while the function of the heart would be changed only marginally. We have found changes in LV, but not in RV: decrease in M3 MR, β1- and β2-adrenoceptor gene expressions that were accompanied by a decrease in MR and BAR receptor binding. No changes were found both in LV systolic and diastolic function as assessed by echocardiography (e.g., similar LV end-systolic and end-diastolic diameter, fractional shortening, mitral flow characteristics, and maximal velocity in LV outflow tract). We have found only marginal changes in specific HR biorhythm parameters. The effects of isoprenaline and propranolol on HR were similar in WT and KO (but with lesser extent). Atropine was not able to increase HR in KO animals. Carbachol decreased the HR in WT but increased HR in KO, suggesting the presence of cardiostimulatory MR. Therefore, we can conclude that although the main cardioinhibitory receptors are not present in the heart, the function is not much affected. As possible mechanisms of almost normal cardiac function, the decreases of both β1- and β2-adrenoceptor gene expression and receptor binding should be considered.
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Acknowledgments
This work was supported by Grant GACR309/09/0406. We thank Dr. Jürgen Wess (NIH, Bethesda, MD, USA) for providing the muscarinic receptor mutant mice, Prof. Michal Zeman (Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia) for advice with biorhythm analysis, and Prof. Ludek Cervenka (Institute for Clinical and Experimental Medicine, Prague, Czech Republic) for the great help with echocardiography study. The technical assistance of Petra Svatosova is greatly acknowledged.
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Jan Benes and Eva Varejkova participated equally on this study.
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Figure S1
Saturation binding to muscarinic (left) and -adrenergic (right) receptors in WT (above) and KO (below) animals. Abscissa: concentration of free radioligand [nmol.l-1].Ordinate: Bmax [fmol.mg prot.-1]. Insert: Scatchard plot of binding. MR Muscarinic receptors; BAR, β-adrenoceptors. (JPEG 75 kb)
Figure S2
Competition binding curves for CGP 20712A (1-AR antagonist) and ICI118.551 (2-AR antagonist) with 3H-CGP12177. Average data from 4 independent experiments. Abscissa: log of antagonist concentration.Ordinate: Bound [% of binding in the absence of antagonist]. (JPEG 49 kb)
Figure S3
The representative echocardiograms in WT and KO mice. Left: WT mice, right: KO mice. Top: Two-dimensional echocardiogram: end diastole, M-mode echocardiogram from the same mouse in the parasternal short-axis view. Middle: left ventricle outflow tract (two-dimensional echocardiogram, pulsed wave Doppler tracing). Bottom: mitral view (two-dimensional echocardiogram, pulsed wave Doppler tracing). (JPEG 129 kb)
Figure S4
The representative figure of spectral density vs. period and spectral density vs. frequency in WT and KO animals. No difference was observed between KO and WT mice. (JPEG 58 kb)
Figure S5
The gene expression of TH, DBH and PNMT in WT and KO animals. No change in gene expression was observed. (JPEG 133 kb)
Figure S6
The changes in activity in KO mice. Top: average activity. Middle: the activity in the light and dark phase (day or night). Bottom: comparison of biorhythm in WT and KO animals. ***p<0.001 different from WT. ###p<0.001 different from KO day, one-way ANOVA (p< 0.0001, F=15.83, df=93). (JPEG 90 kb)
Figure S7
The changes in temperature in KO mice. Top: average temperature. Middle: the temperature in the light and dark phase (day or night). Bottom: comparison of biorhythm in WT and KO animals. ****p<0.0001 different from WT, ***p<0.001 different from WT day, ###p<0.001 different from KO day, +++p<0.001 different from WT night, one-way ANOVA (p< 0.0001, F=36.18, df=93). (JPEG 79 kb)
Figure S8
The actograms of representative WT and KO mice showing increased values of respective parameters (black rectangles). Abscissa: time starting at 6:00 AM and showing two consecutive days in one row. Ordinate: The number of row representing two days. From top to bellow: WT activity, KO activity, WT HR, KO HR, WT temperature, KO temperature. (JPEG 176 kb)
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Benes, J., Varejkova, E., Farar, V. et al. Decrease in heart adrenoceptor gene expression and receptor number as compensatory tool for preserved heart function and biological rhythm in M2 KO animals. Naunyn-Schmiedeberg's Arch Pharmacol 385, 1161–1173 (2012). https://doi.org/10.1007/s00210-012-0800-9
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DOI: https://doi.org/10.1007/s00210-012-0800-9