1. Transgenic overexpression of the β2-adrenergic receptor (β2AR) in mouse heart augments baseline cardiac function in a ligand-independent manner, due to the presence of spontaneously active β2AR (β2AR*). This study aims to elucidate the mechanism of β2AR*-mediated modulation of cardiac excitation-contraction (EC) coupling. 2. Confocal imaging was used to analyse Ca2+ sparks and spatially resolve Ca2+ transients in single ventricular myocytes from transgenic (TG4) and non-transgenic (NTG) littermates. Whole-cell voltage- and current-clamp techniques were used to record L-type CA2+ currents (I(Ca)) and action potentials, respectively. 3. In the absence of any β2AR ligand, TG4 myocytes had greater contraction amplitudes, larger Ca2+ transients and faster relaxation times than did NTG cells. 4. The action potentials of TG4 and NTG myocytes were similar, except for a prolonged endstage repolarization in TG4 cells; the I(Ca) density and kinetics were nearly identical. The relationship between peak Ca2+ and contraction, which reflects myofilament Ca2+ sensitivity, was similar. 5. In TG4 cells, the frequency of Ca2+ sparks (spontaneous or evoked at -40 mV) was 2-7 times greater, despite the absence of change in the resting Ca2+ sarcoplasmic reticulum (SR) Ca2+ content, and I(Ca). Individual sparks were brighter, broader and lasted longer, leading to a 2.3-fold greater signal mass. Thus, changes in both spark frequency and size underlie the greater Ca2+ transient in TG4 cells. 6. The inverse agonist ICI 118,551 (ICI, 5 x 10-7 M), which blocks spontaneous β2AR activation, reversed the aforementioned β2AR* effects on cardiac EC coupling without affecting the sarcolemmal I(Ca). However, ICI failed to detect significant constitutive β2AR activity in NTG cells. 7. We conclude that β2AR*-mediatecl signalling enhances SR release channel activity and Ca2+-induced Ca2+ release in TG4 cardiac myocytes, and that β2AR* enhances EC coupling by reinforcing SR Ca2+ cycling (release and reuptake), but bypassing the sarcolemmal I(Ca).
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