Isolated Spontaneously Beating Atria

Isolated spontaneously beating atria were prepared using a previously described method (Braganca et al., 2016), with some modifications. In brief, hearts were rapidly excised after decapitation followed by exsanguination (Rodent guillotine, Stoelting 51330), and placed in a physiological solution (Tyrode’s solution) composed of (mM): NaCl 137; KCl 4.7; CaCl₂ 1.8; MgCl₂ 1; NaH₂PO₄ 0.4; NaHCO₃ 11.9; glucose 11.2 and gassed with 95% O₂ + 5% CO₂ (at pH 7.4). Hearts were allowed to beat freely for a few seconds at room temperature, to empty its blood content. The paired rat atria with the SAN region were dissected out, cleaned of fatty tissues, and suspended in a 14-ml organ bath containing gassed Tyrode’s solution at 37 ºC. Each auricular appendage was tied and connected with thread to the organ bath wall and to an isometric force transducer (MLT050/D; AD Instruments, Colorado Springs, CO, USA). Changes in isometric tension were recorded continuously using a PowerLab data acquisition system (Chart 5, version 4.2; AD Instruments, Colorado Springs, CO, USA). The preparations were allowed to equilibrate for 30–40 min. During this time, the preparations were continuously superfused with Tyrode’s solution (1 ml/min) and the tension was adjusted to 9.8 mN. This procedure allows atria (with intact SA node) to progressively recover rhythmic spontaneous beatings (average of 247 ± 5 beats min⁻¹ at the beginning of the experimental protocol, n = 79); preparations with spontaneous atrial rate below 200 beats min⁻¹ or exhibiting rhythm variations above 10 beats min⁻¹ during equilibrium were discarded to ensure measurements were made in atria with intact primary pacemaker SAN activity. None of the preparations exhibited noticeable signs of ectopic-activity caused by secondary pacemakers, usually related to asynchronous and abnormal contractions.

Under these experimental conditions, spontaneously beating rat atria respond to muscarinic and β-adrenergic stimulation, but are unaffected by the application of atropine or atenolol alone used in concentrations high enough (1 µM and 3 µM, respectively) to prevent the effects of acetylcholine (100 µM) and isoproterenol (30 nM), respectively (data not shown). Thus, myographic recordings reported in this study include rate (chronotropic effect) and contractile force (inotropic effect) of spontaneously beating atria measured in the absence of cholinergic and/or adrenergic tone.