β4-Adrenoceptors are more effective than β1-adrenoceptors in mediating arrhythmic Ca2+ transients in mouse ventricular myocytes

Abstract

Putative beta4-adrenoceptors mediate cardiostimulation and arrhythmias in mammalian heart. Both beta1- and putative beta4-adrenoceptors mediate arrhythmias but through different mechanisms. To elucidate further the mechanisms of cardiostimulation and arrhythmias we measured Ca2+ transients and L-type Ca2+ currents in mouse ventricular myocytes. We used (-)-CGP 12177, an antagonist of beta1- and beta2-adrenoceptors with agonist properties at the putative beta4-adrenoceptor, and (-)-isoprenaline as an agonist for beta1- and beta2-adrenoceptors. (-)-CGP 12177 increased Ca2+ transients in electrically stimulated cells loaded with Indo-1. The maximum increase of Ca2+ transients caused by (-)-CGP 12177 amounted to approximately one-third of that caused by maximally effective (-)-isoprenaline concentrations. Both (-)-CGP 12177 and (-)-isoprenaline caused concentration-dependent arrhythmic Ca2+ transients. The arrhythmias appeared at paced Ca2+ transients and between paced Ca2+ transients. The arrhythmic potency of (-)-CGP 12177 (-logEC50=9.4) was approximately 40 times greater than that of (-)-isoprenaline (-logEC50=7.8). L-type Ca2+ current was measured in the whole cell configuration of the patch clamp technique. In the presence of both 3-isobutyl 1-methylxanthine (6 micromol/l) and (-)-propranolol (500 nmol/l), (-)-CGP 12177 (100 nmol/l) increased significantly L-type Ca2+ current by 19% of the effect of (-)-isoprenaline. The (-)-CGP 12177-evoked increase of Ca2+ transients contrasts with the smaller effects on L-type Ca2+ current, suggesting that activation of the putative beta4-adrenoceptor causes a more efficient Ca2+-induced Ca2+ release than activation of the beta1-adrenoceptor. Beta4-Adrenoceptors mediate arrhythmias with smaller Ca2+ transients and smaller increases of L-type Ca2+ current than beta1-adrenoceptors, in line with different but still unknown mechanisms as previously suggested for the intact heart.

Abolition of (-)-CGP 12177-evoked cardiostimulation in double ? 1 /? 2 -adrenoceptor knockout mice. Obligatory role of ? 1 -adrenoceptors for putative ? 4 -adrenoceptor pharmacology

“…In wild-type mice (-)-[ 3 H]CGP 12177 labelled a highaffinity (K D~0 .5 nM), relatively low-density binding population (16 fmol/mg protein), consistent with β 1 -AR plus β 2 -AR, similar to that recently observed with (-)-[ 125 I]iodocyanopindolol (11 fmol/mg protein; Heubach et al 1999). As expected, this binding population was absent in the ventricle of β 1 /β 2 -AR double knockout mice.…”

“…Moreover, in murine ventricular myocytes (-)-CGP 12177 causes arrhythmic Ca 2+ transients and is approximately 40 times more potent than (-)-isoprenaline for this effect . These arrhythmogenic effects of (-)-CGP 12177 are resistant to blockade by (-)-propranolol (Lowe et al 1998;Freestone et al 1999;Sarsero et al 1999) but blocked by (-)-bupranolol , which are characteristics of putative β 4 -AR (Kaumann 1989(Kaumann , 1997. The arrhythmic potency of (-)-CGP 12177 is, however, considerably higher than its cardiostimulant potency and matches its affinity for β 1 -AR, suggesting mediation through a highaffinity β 1 -AR state ) that is clearly different from a low-affinity state that mediates positive inotropic and chronotropic effects of (-)-CGP 12177 in a variety of species from mouse ) to man (Kaumann 1996;Kaumann and Molenaar 1997).…”

Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through β1- and putative β4-adrenoceptors in mouse left atria: no evidence for β2- and β3-adrenoceptor function

“…We do not know the density the β 2 -adrenoceptor population in mouse left atrium but the receptors seem irrelevant to left atrial function in this species. β 1 -, β 2 -and putative β 4 -adrenoceptor populations have been detected with binding assays in mouse ventricle (Heubach et al 1999;Kaumann et al 1998) and the density of β 2 -adrenoceptors comprises approximately one-third of the density of β 1 -adrenoceptors (Heubach et al 1999). Interestingly, Xiao et al (1999) reported that the β 2 -adrenoceptor-selective agonist zinterol did not enhance contractions of mouse ventricular myocytes; only after pretreatment with PTX did zinterol cause concentration-dependent increases in myocyte contraction.…”

“…However, there is not yet evidence whether the cardiostimulant effects of (-)-CGP 12177 are mediated through native cardiac β 1 -adrenoceptors and if this were so, it would be a state of the receptor with low affinity for (-)-propranolol and β 1 -adrenoceptorselective CGP 20712A (Kaumann and Molenaar 1996). For further discussion for the alternatives of an atypical β 1 -adrenoceptor state or putative β 4 -adrenoceptor see Lowe et al (1999) and Freestone et al (1999). Regardless of whether we are dealing with an atypical state of the β 1 -adrenoceptor or a putative β 4 -adrenoceptor, it appears that the comparative small signals of cyclic AMP and cyclic AMP-dependent protein kinase observed with (-)-CGP 12177 ) are unlikely to originate from simultaneous coupling of the receptor to G s and G i/o protein.…”

“…We asked whether inactivation of G i/o proteins by PTX modifies or uncovers the function of native β 1 -, β 2 -, β 3 -and putative β 4 -adrenoceptors in mouse atrium, a suitable tissue increasingly used from transgenic mice (Bond et al 1995;Heubach et al 1999;Kaumann et al 1998;Zolk et al 1998). We introduce a procedure consisting in a 24 h in vitro pretreatment of the atria with PTX, followed by pacing of the atria and assessment of inotropic responses to agonists under conditions of selective β-adrenoceptor subtype activation.…”