Amitriptyline functionally antagonizes cardiac H2 histamine receptors in transgenic mice and human atria

Abstract

We have previously shown that histamine (2-(1H-imidazol-4-yl)ethanamine) exerted concentration-dependent positive inotropic effects (PIE) or positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic (H₂R-TG) mice that overexpress the human H₂ histamine receptor (H₂R) in the heart; however, the effects were not seen in their wild-type (WT) littermates. Amitriptyline, which is still a highly prescribed antidepressant drug, was reported to act as antagonist on H₂Rs. Here, we wanted to determine whether the histamine effects in H₂R-TG were antagonized by amitriptyline. Contractile studies were performed on isolated left and right atrial preparations, isolated perfused hearts from H₂R-TG and WT mice and human atrial preparations. Amitriptyline shifted the concentration-dependent PIE of histamine (1 nM-10 μM) to higher concentrations (rightward shift) in left atrial preparations from H₂R-TG. Similarly, in isolated perfused hearts from H₂R-TG and WT mice, histamine increased the contractile parameters and the phosphorylation state of phospholamban (PLB) at serine 16 in the H₂R-TG mice, but not in the WT mice. However, the increases in contractility and PLB phosphorylation were attenuated by the addition of amitriptyline in perfused hearts from H₂R-TG. In isolated electrically stimulated human atria, the PIE of histamine that was applied in increasing concentrations from 1 nM to 10 μM was reduced by 10-μM amitriptyline. In summary, we present functional evidence that amitriptyline also acts as an antagonist of contractility at H₂Rs in H₂R-TG mouse hearts and in the human heart which might in part explain the side effects of amitriptyline.

Keywords: Amitriptyline; Chronotropy; H2-histamine receptor; Histamine; Human atrium; Inotropy; Phospholamban phosphorylation; Transgenic mice.

Fig. 1

Scheme of a cardiomyocyte: histamine can bind to the H₂ histamine receptor in H₂R-TG and human atrium; subsequently, the activity of adenylyl cyclase (AC) is augmented in the sarcolemma via stimulatory G-proteins (G_s); thereafter cAMP increases, and this activates the cAMP-dependent protein kinase (PKA). PKA increases cardiac force generation and relaxation by increasing the phosphorylation state (P) of the L-type Ca²⁺ channel (LTCC), phospholamban (PLB), and other regulatory proteins. Ca²⁺ initiates release of Ca²⁺ from the sarcoplasmic reticulum where it is usually bound to calsequestrin (CSQ) via ryanodine receptors (RYR) into the cytosol, where Ca²⁺ activates myofibrils and leads to increased inotropy. In the diastole, Ca²⁺ is taken up into the sarcoplasmic reticulum via a sarcoplasmic reticulum Ca²⁺ ATPase (SERCA), whose activity is higher when the phosphorylation state of PLB is elevated by PKA. The phosphorylation of proteins is reduced by protein phosphatases (PP). The H₂R can be antagonized by amitriptyline; thus, PLB phosphorylation is not increased, force is not augmented, and relaxation is not hastened. Isoprenaline can stimulate likewise the β-adrenoceptor, which can also be antagonized by amitriptyline

Fig. 2

a Original recording of the force of contraction (FOC) in left atrium from transgenic mice that overexpress the H₂ receptor (H₂R-TG). First, a concentration response curve for histamine is shown; thereafter histamine was washed out, 10-μM amitriptyline was added, and a second concentration response curve for histamine was constructed. Finally, the β-adrenoceptor agonist isoprenaline was added. b Effect of histamine alone (open circles) or in the additional presence of 1-μM amitriptyline (closed circles) or 10-μM amitriptyline (red circles) on the FOC in isolated electrically driven (1 Hz) left atrium of H₂R-TG. Ordinate: increase in force of contraction in relations to the maximum effect of histamine (=100%). Abscissa: logarithm of histamine concentration. ^★indicates first significant difference (P < 0.05) vs. Ctr (= pre-drug value); ^#p < 0.05 versus control w/o amitriptyline

Fig. 3

Left side (a, c, e): effect of histamine alone (open circles) or in the additional presence of 1-μM (a), 3-μM (c), or 10-μM (e) amitriptyline (closed circles) on the maximum rate of force development in isolated electrically driven (1 Hz) left atrium of H₂ histamine receptor overexpressing mice (H₂R-TG). Ordinate in % of maximum change of force development (ΔdF/dt_max). Ctr = basal contraction before drug addition. Right side (b, d, f): effect of histamine alone (open circles) or in the additional presence of 1-μM (b), 3-μM (d), or 10-μM (f) amitriptyline (closed circles) on the minimum rate of force development in isolated electrically driven (1 Hz) left atrium of H₂R-TG mice. Ordinate in % of minimum change of force development (ΔdF/dt_min). Ctr = basal contraction before drug addition. Abscissae: logarithm of histamine concentration. ^★indicates first significant difference (P < 0.05) vs. Ctr; ^#p < 0.05 versus control w/o amitriptyline

Fig. 4

a Effect of histamine alone (open circles) or in the additional presence of 10-μM amitriptyline (closed circles) on the change of shortening in time to peak tension (T_r) in isolated electrically driven (1 Hz) left atrium of H₂ histamine receptor overexpressing mice (H₂R-TG). Ordinate: change in T_r in milliseconds (ms). Ctr = basal T_r before drug addition. b Effect of histamine alone (open circles) or in the additional presence of 10-μM amitriptyline (closed circles) on the change of shortening in time of relaxation (T_f) in isolated electrically driven (1 Hz) left atrium of H₂R-TG mice. Ordinate: change in T_f in milliseconds (ms). Ctr = basal T_f before drug addition. Abscissae: logarithm of histamine concentration. ^★indicates first significant difference (P < 0.05) vs. Ctr; ^#p < 0.05 versus control w/o amitriptyline

Fig. 5

Effect of histamine alone (open circles) or in the presence of 1-μM (closed circles) or 3-μM (red circles) amitriptyline in isolated spontaneously beating right atrium of H₂R-TG. Ordinate: beating rate in beats per minute. Abscissae: logarithm of histamine concentration. ^★indicates first significant difference (P < 0.05) vs. Ctr (= pre-drug value); ^#p < 0.05 versus control w/o amitriptyline

Fig. 6

Western blot analysis of phospholamban (PLB) phosphorylation at serine 16 in Langendorff hearts from H₂R-TG and WT mice perfused with histamine (1 μM) alone or in the combined presence with amitriptyline (10 μM). Calsequestrin (CSQ) was used as loading control. Ordinate: ratio of serine 16 phosphorylation of PLB and CSQ. *p < 0.05 vs indicated group. The numbers in the bars indicate the numbers of experiments. More details are shown in supplementary Fig. 1.

Fig. 7

Effect of histamine alone (control, open circles) or in the additional presence of 10-μM amitriptyline (closed circles) on the force of contraction (FOC) in isolated electrically driven (1 Hz) human atrial preparations. Six preparations from four patients were used. ^★p < 0.05 vs. Ctr (= pre-drug value); ^#p < 0.05 versus control w/o amitriptyline