Arrhythmogenic effects of beta2-adrenergic stimulation in the failing heart are attributable to enhanced sarcoplasmic reticulum Ca load

Abstract

Ventricular tachycardia in heart failure (HF) can initiate by nonreentrant mechanisms such as delayed afterdepolarizations. In an arrhythmogenic rabbit model of HF, we have shown that isoproterenol induces ventricular tachycardia in vivo and aftercontractions and transient inward currents in HF myocytes. To determine whether beta(2)-adrenergic receptor (beta(2)-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies. Intravenous zinterol (2.5 microg/kg) led to ventricular arrhythmias, including ventricular tachycardia up to 13 beats long in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect blocked by beta(2)-AR antagonist ICI-118,551 (0.2 mg/kg). In field-stimulated myocytes (0.5 to 4 Hz, 37 degrees C), beta(2)-AR stimulation (1 micromol/L zinterol+300 nmol/L beta(1)-AR antagonist CGP-29712A) induced aftercontractions and Ca aftertransients in 88% of HF versus 0% of control myocytes (P<0.01). beta(2)-AR stimulation in HF (but not control) myocytes increased Ca transient amplitude (by 29%), sarcoplasmic reticulum (SR) Ca load (by 28%), the rate of [Ca](i) decline (by 28%; n=12, all P<0.05), and phospholamban phosphorylation at Ser16, but Ca current was unchanged. All of these effects in HF myocytes were blocked by ICI-118,551 (100 nmol/L). Although total beta-AR expression was reduced by 47% in HF rabbit left ventricle, beta(2)-AR number was unchanged, indicating more potent beta(2)-AR-dependent SR Ca uptake and arrhythmogenesis in HF. Human HF myocytes showed similar beta(2)-AR-induced aftercontractions, aftertransients, and enhanced Ca transient amplitude, SR Ca load and twitch [Ca](i) decline rate. Thus, beta(2)-AR stimulation is arrhythmogenic in HF, mediated by SR Ca overload-induced spontaneous SR Ca release and aftercontractions.

Figure 1

Spontaneously-occurring ventricular arrhythmias in conscious failing rabbits and aftercontractions in isolated ventricular myocyte in response to bolus injection (2.5μg/kg) and superfusion (1 μM) of zinterol (Zin), respectively. A) Lead II ECG tracings showing sinus rhythm at baseline (left) and runs of VT with zinterol (middle) prevented by pretreatment with the β₂-AR blocker ICI-118,551 (right). B) 3 Hz cell shortening at baseline conditions at 37°C (left) and aftercontractions (ACs) induced by zinterol (middle) prevented by ICI (right).

Figure 2

β₂-stimulation (zinterol + CGP) induces after-Ca-transients in HF myocytes. A) Ca transients (2 Hz) of myocytes from control rabbits before (left) and after zinterol + CGP. B) Baseline Ca transients (2 Hz) of myocytes from failing rabbit (left) and induction of after-transients with zinterol + CGP (right).

Figure 3

Ca transients in control rabbit myocytes are not altered by β₂-stimulation (zinterol + CGP). A) 1 Hz (upper) & 3 Hz (lower) Ca transients and caffeine-induced Ca transient records at baseline (left) and after zinterol + CGP (right). B) mean values (± SEM) of Ca transient amplitude and SR Ca content at 1 Hz (left, n=14) and 3 Hz (right, n=6) at baseline and after perfusion with zinterol + CGP (Zin+CGP). C) Cell shortening at 1 (left, n=7) and 3 Hz (right, n=4) and during rapid caffeine application at baseline and with zinterol + CGP.

Figure 4

Ca transients in HF rabbit myocytes are enhanced by β₂-stimulation (zinterol + CGP), and reversed by the β₂-blocker ICI. A) 1 and 3 Hz Ca transients and SR Ca content records at baseline (left) and with zinterol + CGP (right) in HF rabbit myocytes. B) Average values of Ca transient amplitude and SR Ca content at 1 Hz (left) and 3 Hz (right) at baseline (Base, n= 12), increase after superperfusion with zinterol + CGP (p< 0.05), and reversed with ICI (grey bars, p<0.05). C) Cell shortening at 1 Hz (left, n=7) and 3 Hz (right, n=6) and during rapid caffeine application at baseline, increase with zinterol + CGP (p< 0.05), and reversed with ICI (p<0.05). *denotes p < 0.05.

Figure 5

β₂-stimulation (zinterol + CGP) increases SERCA, but does not alter NCX in HF rabbit myocytes (or either in control). A) Superimposed normalized 1 Hz Ca transients at baseline and with β₂-AR show no difference in control rabbit myocytes (left) but decline faster with zinterol +CGP in HF rabbit myocytes (right). B) Superimposed normalized caffeine-induced Ca transient ± zinterol +CGP show similar decay in control (left) and HF (right) rabbit myocytes. C) Mean values of exponential decay (τ) of twitch Ca transients and caffeine-induced Ca transients at baseline and with zinterol + CGP in nonfailing rabbit myocyte (left, n=11) and HF rabbit myocytes (right, n=12). * denotes p < 0.05.

Figure 6

Zinterol effects on I_Ca on control and HF rabbit myocytes. A) L-type I_Ca at 0 mV (left) and mean I-V curve (right) in control rabbit myocytes ± zinterol (Zin) (n=6). B) I_Ca at 0 mV (left) and I-V curve (right) in HF rabbit myocytes ± zinterol (n=5). * denotes p < 0.05.

Figure 7

PLB phosphorylation and β-AR density. A) Effects of β₂-AR stimulation of PLB phosphorylation at Ser-16 (PKA) and Thr-17 (CaMKII) sites. Representative Western blots (above) and summarized data (below) for freshly-isolated control and HF rabbit myocytes at baseline or after 10 min exposure to zinterol (1 μM) + CGP 20712A (300nM) in the absence or presence of 100nM ICI 118,551 (n= 4 or 5). B) β-AR density in LV homogenates, indicating total β-AR density and ¹²⁵I-CYP affinity (left) and amounts of β₁- and β₂-AR, based on the component sensitive to either 100 nM CGP-20712A (β₁-AR) or 1 μM ICI-118,551 (β₂-AR), and the percent β₂-AR (right).

Figure 8

Human HF myocytes and β₂-stimulation (zinterol + CGP) induce aftertransients, and increase Ca transient amplitude, Ca transient decline and SR Ca load in HF human myocytes (effects reversed by ICI). A) 1 Hz Ca transients and caffeine-induced Ca transient at baseline, with zinterol+CGP showing a spontaneous aftertransient (AT) before caffeine application (middle) and after perfusion with ICI. B) Mean values of Ca transient amplitude and caffeine-induced Ca transient at baseline, which increase with zinterol + CGP (n=6, p<0.05 vs baseline), and reverse with ICI (gray bar, p< 0.05 vs Zin+CGP). C) Superimposed 1 Hz normalized Ca transient records at baseline, showing faster decline with β₂-AR stimulation, and reversal with ICI. D) mean values of exponential decay of 1 Hz Ca transient at baseline, with β₂-AR activation (n=6, p<0.05 vs baseline), and with ICI addition.