Ca2+ dependence of α-adrenergic effects on the contractile properties and Ca2+ homeostasis of cardiac myocytes

Abstract

α-Adrenergic stimulation is known to enhance myocardial contractility. Adult rat left ventricular myocytes bathed in 1 mM [Ca2+] (Ca(o)) and electrically stimulated at 0.2 Hz responded to α-adrenergic stimulation with 50 μM phenylephrine and 1 μM propranolol with an increase in twitch amplitude to 177.1 ± 25.6% of control (mean ± SEM). In contrast, when cell Ca2+ loading was increased by bathing cells in 5 mM Ca(o), α-adrenergic stimulation decreased twitch amplitude to 68.6 ± 8.2% of control. Time-averaged cytosolic [Ca2+] of cells in 1.0 mM Ca(o) is enhanced via an increase in the frequency of electrical stimulation. When myocytes were stimulated at 2 Hz in 1 mM Ca(o), α-adrenergic stimulation did not increase twitch amplitude (103.8 ± 12.4% of control). In myocytes loaded with the Ca2+ probe indo-1, α-adrenergic effects during stimulation at 0.2 Hz (an increase in twitch amplitude in 1 mM Ca(o) and a decrease in twitch amplitude in 5 mM Ca(o)) were associated with similar changes in the indo-1 transient. In 5 mM Ca(o), spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) occurred in the diastolic interval between twitches (2.9 ± 1.4 spontaneous SR Ca2+ oscillations/min; n = 7); α-adrenergic stimulation abolished these oscillations in six of seven cells. Thus, an increase in the frequency of spontaneous diastolic SR Ca2+ release (i.e., Ca2+ overload) is not the mechanism for the negative inotropic effect of α-adrenergic stimulation in 5 mM Ca(o). In experiments with unstimulated myocytes, we determined whether the effect of α-adrenergic stimulation on cell Ca2+ homeostasis and oscillatory SR Ca2+ release observed in 5 mM Ca(o) occurs only during electrical stimulation, when voltage-dependent currents are operative, or also at rest. Unstimulated rat ventricular myocytes in 5 mM Ca(o) exhibit oscillatory SR Ca2+ release; α-adrenergic stimulation decreased the frequency of these oscillations to 53.9 ± 8.9% of control, and this effect was blocked by 1 μM prazosin. In unstimulated indo-1-loaded myocytes α-adrenergic stimulation decreased the resting indo-1 fluorescence ratio in 5 mM Ca(o), whereas it had no effect in 1 mM Ca(o). Additional experiments were aimed at defining a role for Ca2+-activated, phospholipid-dependent protein kinase C (PKC) for the negative inotropic effect of α-adrenergic stimulation in 5 mM Ca(o). Short-term preexposure to 0.1 μM 4β-phorbol 12-myristate 13-acetate (PMA) has been shown to maximally activate PKC. PMA, in 5 mM Ca(o), decreased contraction and indo-1 transient amplitudes and prevented any additional negative inotropic action by α-adrenergic stimulation. Additionally, staurosporine (5 nM), a PKC inhibitor, abolished the α-adrenergic-stimulated decrease in twitch amplitude in 5 mM Ca(o). Thus, an increase in cytosolic [Ca2+] achieved via an increase in Ca(o) or by rapid pacing leads to effects of α-adrenergic stimulation on the contractile properties and Ca2+ homeostasis of rat ventricular myocytes that are different from those observed in lower Ca(o). These results may be explained with a [Ca2+]-dependent enhancement of the activity of PKC, an enzyme that is activated during α-adrenergic stimulation of the myocardium.