P119Beta-2 adrenergic receptor signaling in adult rat ventricular myocytes at 4, 8 and 16 weeks after myocardial infarction

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Abstract

We have previously shown that β2AR-induced cAMP signal localisation is disrupted at 16 weeks post-myocardial infarction in a rat model of heart failure. In this study we aimed to investigate the changes in β2AR signaling as failure develops. Chronic myocardial infarction (MI) was induced by permanent coronary arterial ligation in adult rats. Adult rat ventricular myocytes (ARVM) were isolated at 4, 8 and 16 weeks after MI. A video-microscope technique (IonOptix) was used to assess contraction of ARVM derived from the ventricular apex. Selective β2AR stimulation was achieved with 1μM Isoproterenol whilst inhibiting β1AR with 300nM CGP20712A. PTX was used to assess the contribution of Gi in the signalling. Cells were transfected with a cAMP sensor construct EPAC2 and cultured for 48 hours for FRET. These were stimulated with 100nM ISO in the presence of 100nM CGP and then further stimulated with Forskolin analogue NKH477 at 5 μM.

ARVMs were hypertrophied at 4, 8 and 16 weeks post-MI. Contraction amplitude (%shortening) was increased compared to control at all 3 time points (Control 3.91 ± 0.5 % n=21; 4 Week 6.70 ± 0.60 n=20 p≤0.001; 8 Week 7.95 ± 0.70 N=9 p≤0.001; 7.89 ± 0.39 N=48 p≤0.001).

In control cells both contractility and cAMP FRET response increased appropriately following β2AR stimulation (Contraction: CGP Baseline 3.34% ± 0.51 N=11 vs. CGP + ISO 7.09% ± 1.13 N=11 p≤0.001). At 4 weeks, contraction was unresponsive to β2AR stimulation but FRET response was normal (expressed as a percentage of maximal AC stimulation after 5 μM NKH477: Control 39.3 ± 2.2% N=42 vs. 35.3% ± 1.5 N=12 p=NS) . ARVMs at 8 weeks had an increased ability of β2AR stimulation to promote cAMP production (Control 39.3% ± 2.2% N=42 vs. 8 Week 53.2 ± 5.0% N=9 p≤0.01). However, contraction was again unresponsive to β2AR stimulation. At 16 Weeks post MI cells did not increase cAMP production upon β2AR stimulation but did increase contraction amplitude (CGP Baseline 8.1 ± 0.75 N=17 vs. CGP + ISO 10.42 ± 1.1 N=17 p≤0.001). PTX pre-treatment produced only modest increases in β2AR-mediated contraction at 8 and 16 weeks, without complete rescue.

Increased basal contraction of surviving myocytes from the infarcted ventricle is consistent with a compensatory hypertrophic phase. Disconnect between cAMP responses and contractile effects suggest that both non-cAMP dependent positive inotropic and concurrent negative inotropic signalling are activated through β2ARs. The lack of rescue by PTX indicates a minor role for Gi coupling in the negative responses. β2AR coupling is dynamically regulated through the development of heart failure.

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