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Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.

To determine the direct functional significance of the beta-adrenergic receptor (AR) kinase 1 (beta ARK1) on myocardial performance in the absence of tonic sympathoadrenal neural activation and mechanical loading, we measured the contractile responses to acute beta 1-AR stimulation in left ventricular myocytes isolated from nontransgenic control (NTG) and transgenic mice overexpressing either beta ARK1 (TG beta K12) or a beta ARK1 inhibitor (TGMini27). Contractile response to five concentrations (10(-8)-10(-7) M) of the beta 1-AR agonist norepinephrine (NE) plus prazosin (10(-6) M) was measured after a 60-s rest, i.e., rested-state contraction (RSC), and during steady-state contraction (SSC) stimulation at 0.5 Hz (23 degrees C). At baseline, resting cell length was significantly greater in TG beta K12 myocytes (P < 0.05); however, there were no significant differences in RSC or SSC among NTG, TG beta K12, or TG Mini27 mice. On the other hand, both the dose-response curve and kinetics for the NE-induced SSC response normalized to RSC (SSC/RSC) were significantly different among experimental groups (P < 0.001). Specifically, maximal SSC induced by NE in myocytes isolated from TG beta K12 was only 70% of the response observed in NTG cells and 50% of the response measured in TGMini27. These data suggest that 1) in the absence of circulating catecholamines or basal sympathetic tone, beta ARK1 actions in single myocytes are minimal, and 2) substantial functional beta ARK1 modulation of beta 1-AR signaling occurs in cardiac myocytes even during short-term beta 1-AR stimulation. These results are consistent with a role for agonist-induced phosphorylation and desensitization of cardiac beta 1-ARs by beta ARK1 in single myocytes and highlight the potential functional importance of beta ARK1 as a critical determinant of the cardiac beta 1-AR contractile response.

Ca2+ sparks, the elementary units of sarcoplasmic reticulum (SR) Ca2+ release in cardiac, smooth and skeletal muscle are localized (2-4 microns ) increases in intracellular Ca2+ concentration, [Ca2+]i, that last briefly (30-100 ms). These Ca2+ sparks arise from the openings of a single SR Ca2+ release channel (ryanodine receptor, RyR) or a few RyRs acting in concert. In heart muscle, Ca2+ sparks can occur spontaneously in quiescent cells at a low rate (100 s-1 per cell). Identical Ca2+ sparks are also triggered by depolarization because the voltage-gated sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) locally increase [Ca2+]i and thereby activate the RyRs by Ca(2+)-induced Ca2+ release (CICR). The exquisite responsiveness of this process, reflected by the ability of even a single DHPR to activate a Ca2+ spark, is perhaps due to the large local increase in [Ca2+]i in the vicinity of the RyR that is a consequence of the close apposition of the DHPRs and the RyRs. In this review we examine our current understanding of cardiac excitation-contraction (EC) coupling in light of recent studies on the elementary Ca2+ release events or Ca2+ sparks. In addition, we further characterized Ca2+ spark properties in rat and mouse heart cells. Specifically we have determined that: (i) Ca2+ sparks occur at the junctions between the transverse-tubules and the SR in both species; (ii) Ca2+ sparks are asymmetric, being 18% longer in the longitudinal direction than in the transverse direction; and (iii) Ca2+ sparks individually do not produce measurable sarcomere shortening (< 1%). These results are discussed with respect to local activation of the RyRs, the stability of CICR, Ca2+ diffusion, and the theory of EC coupling.

Intracellular calcium ([Ca2+]i) elevation may mediate cardiac arrhythmias. However, direct measurement of the rapid alterations of [Ca2+]i on a beat-to-beat basis using fast temporal resolution and without signal averaging in the spontaneously beating in vivo heart is lacking. Furthermore, data from an isolated spontaneously beating myocyte preparation that develops arrhythmia similar to that in the in vivo heart are unavailable. We measured rapid changes of [Ca2+]i with fast temporal resolution in isolated spontaneously beating neonatal rat ventricular myocytes with cell-to-cell communication and characterized the interrelation between [Ca2+]i and arrhythmia. An elevated extracellular calcium ([Ca2+]o) concentration of 10.8 mM induced premature beats, a rapid beating rate (tachyarrhythmia), and chaotic or fibrillatory beating activity in a small group of myocytes. [Ca2+]i levels during systole increased from the nanomolar to micromolar concentration range before arrhythmia development. Spontaneous oscillations of [Ca2+]i during diastole could evoke a spontaneous tachyarrhythmia. In the presence of [Ca2+]i elevation, a spontaneous tachyarrhythmia could induce severe [Ca2+]i overload. Reduction of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid AM (5 microM) in the presence of 10.8 mM [Ca2+]o reversed the arrhythmia. In single ventricular myocytes superfused with 10.8 mM [Ca2+]o, oscillations of membrane potential characteristic of transient inward current occurred that were prevented by ryanodine (0.1 microM), an inhibitor of Ca2+ flux across the sarcoplasmic reticulum. This study characterizes 1) an isolated multicellular myocyte model of arrhythmia similar to that evident in in vivo hearts, 2) elevation of [Ca2+]i with systolic [Ca2+]i levels of 1-3 microM and diastolic [Ca2+]i oscillations before the initiation of arrhythmia, 3) tachyarrhythmia as a cause of severe [Ca2+]i overload, which may be important in the perpetuation and degeneration of arrhythmias, and 4) reversal of arrhythmia with reduction of [Ca2+]i. The results in the isolated myocyte model may have relevance to the generation and perpetuation of certain cardiac arrhythmias associated with calcium overload.