High levels of circulating epinephrine trigger apical cardiodepression in a β2-adrenergic receptor/Gi-dependent manner: a new model of Takotsubo cardiomyopathy

Abstract

Background: Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic β(2)-adrenergic receptor (β(2)AR) from canonical stimulatory G-protein-activated cardiostimulant to inhibitory G-protein-activated cardiodepressant pathways.

Methods and results: We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G(i) inactivation by pertussis toxin pretreatment. β(2)AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a β(2)AR-Gi-dependent manner. Preventing epinephrine-G(i) effects increased mortality in the Takotsubo model, whereas β-blockers that activate β(2)AR-G(i) exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality.

Conclusions: We suggest that biased agonism of epinephrine for β(2)AR-G(s) at low concentrations and for G(i) at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in β(2)ARs explaining the differential regional responses. We suggest this epinephrine-specific β(2)AR-G(i) signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

Figure 1

Takotsubo cardiomyopathy is epinephrine-specific. Effects of 4.28x10^-8 moles.100g^-1 epinephrine (dark red bars) and 1.43x10^-7moles.100g^-1 norepinephrine (dark blue bars) on apical (A), mid left-ventricular (B) and basal myocardium contractility (C). Values are expressed as the mean percentage change in LV fractional shortening (%∆FS) from baseline (untreated) levels ± SEM at each 5 min time point following injection. N=6 (epinephrine), n=6 (norepinephrine) (**p<0.01, ***p<0.001, ****p<0.0001 vs baseline FS = 0). Abbreviation: B (baseline). RM ANOVA: epinephrine vs norepinephrine: p<0.001 (apex), p<0.001 (MLV), p=NS (base); Time: P<0.001 (apex), P<0.001 (MLV), P<0.05 (base).

Figure 2

In vivo Takotsubo Cardiomyopathy model and prevention by Pertussis toxin pretreatment. Contractile responses after an intravenous bolus injection of epinephrine (4.28x10^-8 moles.100g^-1-dark red bars) on left ventricular apical (A), mid left-ventricular (B) and basal myocardium (C). Values are expressed as the mean percentage change in LV fractional shortening (%∆FS) from baseline (untreated) levels ± SEM at each 5 minute time point following injection. Light blue bars show time-matched inotropic responses of the apical, mid-left ventricular and basal myocardium in PTX (25µg.Kg^-1) pre-treated animals after equivalent i.v. epinephrine bolus, with loss of apical and MLV hypokinesis. N=6 (control epinephrine), n=5 (epinephrine+PTX) (**p<0.01, ***p<0.001 vs baseline FS = 0). Abbreviations: B (baseline). RM ANOVA (epinephrine vs epinephrine+PTX): p<0.001 (apex), p<0.01 (MLV), p<0.05 (base); Time: P<0.001 (apex), P<0.001 (MLV), P<0.001 (base).

Figure 3

In vitro Takotsubo cardiomyopathy model induced by high dose epinephrine exposure. Effect of 20 min pretreatment with epinephrine (Epi-pre, 1µM), followed by 10 min wash, on subsequent β₂AR contractile (A) and cAMP (B) responses with Gi (PTX-sensitive) component. A, Contraction amplitude in isolated rat ventricular myocytes: peak fold increase over basal: Control (n=15); Epi-pretreated alone (n=15); Epi-pretreated +PTX (n=7). B, Whole cell cyclic AMP levels, measured using an EPAC2-FRET sensor. Control (n=40); Epi-pretreated alone (n=10); Epi-pretreated +PTX (n=9). *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA Kruskal-Wallis test. C, β₂AR-mediated inotropic response to 100nM isoproterenol in the presence of the β₁AR blocker CGP20712A (300nM), peak fold increase over basal in control (n=13) or PTX-treated rat ventricular myocytes (n=13), in the presence and absence of SB20380 2.5μM. **P<0.01, ***P<0.001 vs control, unpaired t-test. D and E, Apically-derived cardiomyocytes demonstrate increased β₂AR levels and responses. D, Proportion of β₂ARs with respect to total βAR radioligand binding in ventricular myocytes from the apex and base of normal rat heart. N=4 preparations, **P<0.01 vs base, paired t-test. E, Apical cardiomyocytes (purple bars) show a larger increase in percentage cell shortening through the β₂AR compared to basal cardiomyocytes (green bars). Fold increase in shortening with 100nM isoproterenol + 300nM CGP20712A. (*p<0.05 apex vs base, paired t-test. Base: n=13 cells; Apex: n=13 cells, n=13 animals).

Figure 4

Epinephrine-mediated β₂AR-Gi signalling is cardioprotective. A, Mortality with in vivo bolus epinephrine (4.28x10^-8 moles.100g^-1) in the absence (n=14) or presence of 0.1-10mg.Kg^-1 SB203580 (n=9), 1mg.Kg^-1 ICI 118,551 (n=5), 1.43x10^-11 moles.100g^-1 propranolol (n=9), 1.43x10^-11 moles.100g^-1 carvedilol (n=12), 4.7 μg/kg/min levosimendan (n=5). *P<0.05 vs epinephrine alone, Fisher’s exact test. B, Survival of adult rat ventricular myocytes (% remaining at 48h compared to time 0) after exposure to 1μM isoproterenol (ISO) in the presence (light blue bars) and absence (red bars) of the β₁AR blocker CGP20712A (300nM), compared to untreated controls (white bars). Myocytes were transduced using adenoviral vectors with GFP (control), the wild-type β₂AR and β₂AR with mutations at the PKA phosphorylation sites 261, 262, 345, 346 S/A (β₂AR-PKA-KO) to prevent switching to Gi. N=6, # P<0.05 vs con/GFP, *p<0.05 vs GFP+ISO, One-way ANOVA. C, Effect of Gi expression upon ISO-induced myocyte toxicity over 48hrs in culture. Myocytes were transduced using adenoviral vectors with GFP (control), or Gi-GFP (Gi) at Day 0. N=6 preparations, *P<0.05, **P<0.01 vs respective control, #P<0.05, ##P<0.01 vs ISO alone, one-way ANOVA.

Figure 5

Agonist-independent negative inotropic effect of betablockers and potentiation of Takotsubo cardiomyopathy. A, Negative inotropic effect of βAR blockers on contraction of ventricular myocytes from failing human heart. Contraction amplitude relative to basal (open bar) for ICI 118,551 (3μM, n=21), propranolol (Prop, 5μM, n=9) and carvedilol (Carv, 3μM, n=24), *P<0.05, ***P<0.001 vs 100%, one-way ANOVA. B and C, The β-blockers propranolol (B) and carvedilol (C) (both 1.43x10^-11 moles.100g^-1 (i.v.)) either enhance or fail to prevent the negative inotropic effects of epinephrine (4.28x10^-8 moles.100g^-1 (i.v.)) at the apex and also reverse the positive effects of epinephrine at the base, in the in vivo rat model. Values are expressed as the mean percentage change in LV FS from baseline (untreated) levels ± SEM at each 5 min point following intravenous injection. N=6 (epi), n=6 (epi+propranolol), n=7 (epi+carvedilol). (**p<0.01, ***p<0.001, ****p<0.0001 vs baseline FS = 0). RM ANOVA epi vs epi+Propranolol: apex, P=0.05; base P<0.01: time, apex P<0.001; base P<0.001. Epi vs epi+carvedilol: apex P=NS; base P<0.001. Time: apex P<0.001; base P<0.001.

Figure 6

Levosimendan rescues the Takotsubo cardiomyopathy model. Effects of 0.28mg/kg/h (4.7 μg/kg/min) levosimendan infusion (i.v.) (black bars) on the inotropic responses of the apical (A), mid left-ventricular (B) and basal myocardium contractility (C) after 4.26x10^-8 moles.100g^-1 epinephrine (i.v.), compared to epinephrine alone (grey bars). Values are expressed as the mean percentage change in LV FS from baseline ± SEM at each 5 min time point following injection. N=6 (epinephrine), n=5 (levosimendan + epinephrine) (**p<0.01, ***p<0.001, ****p<0.0001 vs baseline FS = 0). RM ANOVA Epi vs epi+levosimendan: P<0.01 (apex), P<0.01 (MLV), P=NS (base). Time: P<0.001 (apex); P<0.001 (MLV); P<0.001 (base).