Localized cAMP-dependent signaling mediates β₂-adrenergic modulation of cardiac excitation-contraction coupling

Recent studies have shown that β²-adrenergic receptor (β₂-AR)- stimulated increases in the intracellular Ca²⁺ (Ca(i)) transient and contraction in cardiac myocytes are dissociated from the increase in adenosine 3',5'-cyclic monophosphate (cAMP) level and are not accompanied by an increase in phospholamban phosphorylation, an acceleration in relaxation, or a reduction in myofilament Ca²⁺ response. Thus we hypothesized that the β₂-AR modulation of cardiac excitation-contraction (EC) coupling may be mediated by either a cAMP-independent mechanism or a compartmentalized cAMP pathway. To directly distinguish between these two possibilities, the responses of the L-type Ca²⁺ current (I(ca)), Ca(i) transient, and contraction to β₂-AR as well as to β₁-AR stimulation were examined in rat ventricular myocytes in the presence or absence of specific inhibitory cAMP analogs, Rp diastereomers of adenosine 3',5'-cyclic monophosphothioate (Rp- cAMPS) and 8-(4-chlorophenylthio)-cAMP (RpCPT-cAMPS). As expected, the positive inotropic effect induced by an adenylyl cyclase activator, forskolin (2 x 10^-7 M), or a β₁-AR agonist, norepinephrine (5 x 10^-8 M) plus prazosin (10^-6 M), was completely blocked by Rp-CPT-cAMPS. More importantly, the responses of I(ca), Ca(i) transient, and contraction to β₂-AR stimulation by zinterol (10^-5 M) or isoproterenol plus a selective β₁-AR antagonist, CGP-20712A, were also entirely abolished by Rp-cAMPS (in the patch-pipette solution) or Rp-CPT-cAMPS (in the bath solution). In pertussis toxin-treated cells, although the response of cAMP was not altered, the β₂-AR-stimulated increase in contraction amplitude was markedly enhanced and accompanied by a hastened relaxation, resulting in a tight association between cAMP and contraction. These results indicate that β₂- AR modulation of cardiac excitation-contraction coupling requires cAMP. The dissociation of β₂-AR-stimulated cAMP production and regulation of myofilament and sarcoplasmic reticulum functions is attributable to a functional compartmentation of the cAMP-dependent signaling due to an activation of β₂-AR-coupled G(i) and/or G(o).