During the development of the murine heart, a variety of electrophysiological changes takes place: both the levels of expression of ion channels and their regulatory properties are altered (Kojima et al. 1990; Maki et al. 1996). During early murine embryogenesis, it has been shown that the L-type Ca2+ channel current (ICa) plays a dominant role in excitation, whereas the slow component of the delayed rectifier K+ channel current (IK,s) is not so apparent until the later stages of embryonic development, or even after birth (Honore et al. 1991; Davies et al. 1995). Also, during development of the mouse heart, the β-adrenergic signalling pathway has been found to be functionally incomplete until the later stages of embryogenesis suggesting a possible link between this pathway and expression of IK,s (Chen et al. 1982; Kojima et al. 1990; An et al. 1996).
The influence of catecholamines acting through β-adrenergic receptors (β-ARs) on the activity and expression of ion channels is of particular interest in the heart as it is well known that in chronic heart failure the myocardial β-AR system is defective. This functional impairment is associated with a decrease in agonist-induced inotropy, and is thought to be caused by a receptor defect, since the adenylyl cyclase response remains intact (Bristow et al. 1982). Specifically, it has been found that there is a selective downregulation of β1-ARs which increases substantially the percentage of total β-ARs that are of the β2-subtype (Bristow et al. 1986; Ungerer et al. 1993). Additionally, a population of remaining receptors (both β1- and β2-ARs) is functionally uncoupled, possibly due to increased homologous desensitization as the levels of β-adrenergic receptor kinase are increased in heart failure (Ungerer et al. 1993). Thus, β-AR agonists used to treat heart failure are not effective chronically and patients are at a higher risk of mortality as a result of the elevated levels of catecholamines (Ginsburg et al. 1983).
Recently, a transgenic mouse model has been developed in which β2-ARs are overexpressed specifically in the heart (Milano et al. 1994). In the hearts of the adult transgenic mice, there is a > 100-fold increase in β2-AR density accompanied by apparent maximal heart rate and cardiac contractility. The physiological changes in heart rate and contractility are not believed to arise simply as a result of stimulation of the increased number of β2-ARs by circulating catecholamines. Instead, they are thought to be due specifically to an increase in the number of β2-ARs present in the active conformation, which are able to activate adenylyl cyclase in the absence of agonist (Milano et al. 1994). This novel transgenic model provides a unique opportunity to investigate the effects of the β-AR pathway on the expression of ionic channels in the heart during development and to determine directly whether increased numbers of receptors by themselves activate functionally relevant steps in the β-AR signal cascade. In addition, it affords an opportunity to study modulatory properties that may be unique to the β2-AR and hence relevant to heart failure when the relative importance of this receptor subtype increases.
Here, we used this mouse model to study the effects of overexpression of the β2-AR on ICa and IK,s in the developing mouse heart. This study had multiple goals. First, we wanted to determine directly whether β2-AR overexpression modulates ion channel activity in the developing mouse heart in the absence of exogenous agonist and whether the effects of β2-AR overexpression could be detected functionally during embryonic development. Second, we used this model to test directly for an inter-relationship between the β2-AR signalling pathway and expression of IK,s. Our results indicate that β2-AR overexpression enhances ICa in a cAMP-dependent manner as early as day 14 of embryogenesis (E14). Surprisingly however, despite a robust sensitivity to exogenous 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), IK,s was not enhanced at any stage of development in β2-AR transgenic positive (TG+) animals. These results clearly indicate unique electrophysiological consequences of β2-AR-induced liberation of cAMP, and are consistent both with compartmentalization of β2-AR-controlled cAMP and distinct localization of L-type Ca2+ and slow delayed rectifier K+ channels.