Norepinephrine-Induced Adrenergic Activation Strikingly Increased the Atrial Fibrillation Duration through β1- and α1-Adrenergic Receptor-Mediated Signaling in Mice

Abstract

Background: Atrial fibrillation (AF) is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals.

Objectives: Our aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR) subtypes, which may be involved in the onset and duration of AF.

Methods and results: Trans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0 ± 8.1 sec). We found that adrenergic activation by intraperitoneal norepinephrine (NE) injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold (656.2 ± 104.8 sec; P<0.001). In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors' effects on AF, we assessed the SR Ca(2+) leak, a major trigger of AF, and consequent spontaneous SR Ca(2+) release (SCR) in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca(2+) leak and SCR. These findings suggest that both β1-AR and α1-AR may play important roles in the development of AF.

Conclusions: We have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments.

Fig 1. Induction of AF by transesophageal atrial burst pacing in mice.

Representative lead II body surface electrocardiogram (ECG) recordings (A). Simultaneous recordings of Lead II body surface ECG (upper) and esophageal ECG (lower) (B-D). (A) AF was induced by transesophageal atrial burst pacing (BP). An AF lasted about 32 seconds before spontaneous conversion into normal sinus rhythm (NSR). (B) Spontaneous conversion from AF to NSR. (C) Representative example of AF episode with disorganized fibrillatory atrial activities and irregular ventricular responses. (D) Conversion from AF to Afl with 4:1 atrioventricular-nodal conduction. Asterisks, arrows and circles indicate P-waves, atrial- and ventricular-electrograms, respectively. All R-R intervals are expressed in milliseconds.

Fig 2. Norepinephrine strikingly elongates the duration of AF.

(A) Schematic diagrams of experimental protocol to induce AF after sympathetic activation in mice. Mice were treated with 1.5 mg/kg of norepinephrine (NE) by intraperitoneal injection followed by transesophageal atrial burst pacing to induce AF. The rectangle represents the period from the start of burst pacing to the termination of AF. Note that, for each individual animal, the longest duration among 10 trials was taken to be the duration of AF after NE administration. (B) The duration of AF was strikingly increased after NE administration in a dose-dependent manner. NE was intraperitoneally injected into each mouse at one of several doses as indicated below, and was followed by transesophageal atrial burst pacing (n = 6–8, *P<0.05 vs CTRL, †P<0.05 vs 2 μg/kg, ‡P<0.05 vs 500 μg/kg) (n = 6–8, ***P<0.001 vs CTRL, †††P<0.001 vs 2 μg/kg, ‡‡‡P<0.001 vs 500 μg/kg, §§P<0.01 vs 1000 μg/kg).

Fig 3. Norepinephrine elongates AF duration through β1- and α1-adrenergic receptor-mediated signaling.

(A) 2 mg/kg of metoprolol, (B) 1 mg/kg of prazosin or natural saline (CTRL) was intraperitoneally injected into conscious mice 45 min before the administration of NE (1.5 mg/kg). Both metoprolol and prazosin treatment significantly shortened the NE-elongated AF. (n = 10–13, *P<0.05, #P<0.1 vs CTRL) (n = 10–15, *P<0.05).

Fig 4. Measurement of Ca²⁺ transient in atrial myocytes.

Representative Ca²⁺ traces of atrial myocytes in the absence (A) or presence (B) of 1 μM NE. Fluo-4 loaded myocytes were electrically paced at 1 Hz for 15 seconds followed by a rapid switch of the extracellular solution from normal Tyrode to 0Na⁺/0Ca²⁺ Tyrode. The spontaneous Ca²⁺ release (SCR) was counted for 30 seconds. The diastolic Ca²⁺ leak from sarcoplasmic reticulum (SR), SR Ca²⁺ leak, was estimated by measuring the downward shift in fluorescence after 1 mM tetracaine treatment. Finally, 10 mM caffeine was administered rapidly to estimate the SR Ca²⁺ content.

Fig 5. Norepinephrine induces SR Ca²⁺ leak and spontaneous Ca²⁺ releases via β1- and α1-AR mediated signaling.

Quantification of spontaneous Ca²⁺ release (A) and SR Ca²⁺ leak (B). Atrial myocytes were incubated with metoprolol or prazosin in the absence (-) or presence (+) of 1 μM NE. Metoprolol and prazosin significantly reversed the NE-enhanced SCR (n = 14–22, **P<0.01, ***P<0.001) and SR Ca²⁺ leak (n = 14–22, *P<0.05). The magnitude of SR Ca²⁺ leak is expressed as a relative value to SR Ca²⁺ content. Values under the graph represent the concentrations of adrenergic receptor antagonists (μM).