Up-regulation and functional effect of cardiac β3-adrenoreceptors in alcoholic monkeys

Abstract

Background: Recent studies link altered cardiac beta-adrenergic receptor (AR) signaling to the pathology of alcoholic cardiomyopathy (ACM). However, the alteration and functional effect of beta(3)-AR activation in ACM are unknown. We tested the hypothesis that chronic alcohol intake causes an up-regulation of cardiac beta(3)-AR, which exacerbates myocyte dysfunction and impairs calcium regulation, thereby directly contributing to the progression of ACM.

Methods: We compared myocyte beta(3)- and beta(1)-AR expression and myocyte contractile ([Ca(2+)](i)), transient ([Ca(2+)](iT)), and Ca(2+) current (I(Ca,L)) responses to beta- and beta(3)-AR stimulation in myocytes obtained from left ventricle (LV) tissue samples obtained from 10 normal control (C) and 16 monkeys with self-administered alcohol for 12 months prior to necropsy: 6 moderate (M) and 10 heavy (H) drinkers with group average alcohol intakes of 1.5 +/- 0.2 and 3.3 +/- 0.2 g/kg/d, respectively.

Results: Compared with control myocytes (C), in alcoholic cardiomyocytes, basal cell contraction (dL/dt(max), -39%, H: 69.8 vs. C: 114.6 microm/s), relaxation (dR/dt(max), -37%, 58.2 vs. 92.9 microm/s), [Ca(2+)](iT) (-34%, 0.23 vs. 0.35), and I(Ca,L) (-25%, 4.8 vs. 6.4pA/pF) were all significantly reduced. Compared with controls, in moderate and heavy drinkers, beta(1)-AR protein levels decreased by 23% and 42%, but beta(3)-AR protein increased by 46% and 85%, respectively. These changes were associated with altered myocyte functional responses to beta-AR agonist, isoproterenol (ISO), and beta(3)-AR agonist, BRL-37344 (BRL). Compared with controls, in alcoholic myocytes, ISO (10(-8) M) produced significantly smaller increases in dL/dt(max) (H: 40% vs. C: 71%), dR/dt(max) (37% vs. 52%), [Ca(2+)](iT) (17% vs. 37%), and I(Ca,L) (17% vs. 27%), but BRL (10(-8) M) produced a significantly greater decrease in dL/dt(max) (H: -23% vs. C: -11%), [Ca(2+)](iT) (-30% vs. -11%), and I(Ca,L) (-28% vs. -17%).

Conclusions: Chronic alcohol consumption down-regulates cardiac beta(1)- and up-regulates beta(3)-ARs, contributing to the abnormal response to catecholamines in ACM. The up-regulation of cardiac beta(3)-AR signaling enhances inhibition of LV myocyte contraction and relaxation and exacerbates the dysfunctional [Ca(2+)](i) regulation and, thus, may precede the development of ACM.

Figure 1

Examples of analog recordings on LV myocyte (A) contractile functional performance and the peak [Ca²⁺]_i transient and (B) I_Ca,L responses obtained from one normal control monkey and one monkey receiving heavy alcohol intake following the 12-month protocol. Compared with control, after 12 months of alcohol intake, myocyte baseline contraction and relaxation (measured as dL/dt_max and dR/dt_max) as well as peak systolic [Ca²⁺]_iT and I_Ca,L were markedly reduced, indicating LV myocyte contractile dysfunction and [Ca²⁺]_i homeostasis impairment.

Figure 2

Examples of superimposed tracings of analog recordings on myocyte contractile functional performance and [Ca²⁺]_iT responses to ISO. Myocytes isolated from the heart tissue of LV obtained by biopsy from one normal and one heavy drinker monkey at the end of the 12-month study period. Myocytes were superfused with HBS solution at 22°C. Myocyte contraction and relaxation were measured as SA, dL/dt_max, and dR/dt_max. Compared with controls, after 12-months of alcohol intake, SA, dL/dt_max, dR/dt_max, and [Ca²⁺]_iT were markedly reduced. In the control myocytes, superfusion of ISO (10⁻⁸ M) caused positive inotropic responses with markedly increased SA, dL/dt_max, dR/dt_max, and [Ca²⁺]_iT. In alcoholic myocytes, the ISO-induced positive action was attenuated.

Figure 3

Examples of superimposed tracings of analog recordings on myocyte contractile functional performance and I_Ca,L responses to BRL. Isolated LV myocytes obtained from one normal and one heavy drinker monkey at the end of the 12-month study period. BRL caused a negative inotropic effect with markedly decreased dL/dt_max dR/dt_max, and I_Ca,L in both normal and alcoholic myocytes. Of note, BRL had a greater effect in decreasing myocyte contraction, relaxation, and I_Ca,L in the alcoholic LV myocytes, indicating myocyte contractile dysfunction and [Ca²⁺]_i homeostasis impairment as presented in ACM exacerbated by BRL.

Figure 4

Group mean (± SD) of β-AR agonist, isoproterenol (ISO), β₃-AR agonist, BRL-37,344 (BRL), and β₃-AR antagonist, L-748,337 (β₃-ANT)-induced changes of myocyte contraction (measured as the velocity of shortening, dL/dt_max) and [Ca²⁺]_iT in normal control and chronic heavy alcohol intake monkeys. Compared with baselines, both dL/dt_max and [Ca²⁺]_iT were significantly increased during ISO superfusion, but significantly decreased with BRL in both normal and alcoholic myocytes. Of note, alcoholic myocytes showed significantly attenuated positive responses to ISO, but enhanced negative responses to BRL. In normal myocytes, β₃-ANT caused no marked changes in dL/dt_max and [Ca²⁺]_iT; by contrast, in alcohol myocytes, β₃-ANT significantly increased dL/dt_max and [Ca²⁺]_IT.

Figure 5

Representative superimposed tracings of analog recordings of contractile response to BRL in both normal and alcoholic myocytes after pretreatment with L-NAME (A) and PTX (B). After pretreatment of myocytes with L-NAME, BRL (10⁻⁸ M)-induced decreases in SA and dL/dt_max were greatly reduced. However, the BRL still markedly decreased SA and dL/dt_max in both normal and alcoholic myocytes. In contrast, pretreated myocytes with PTX prevented BRL-caused changes in both normal and alcoholic myocytes, indicating that β₃-AR activation is coupled with G_i pathways.

Figure 6

LV myocyte β₃-AR mRNA expression following 12-month protocol. RT-PCR analysis of β₃-AR mRNA expression in the normal (line 1) and alcoholic (lines 2 and 3) myocytes obtained from one normal control, one moderate (M), and one heavy drinker (H) monkey. Of the LV myocyte total RNA samples examined with RT-PCR, β₃-AR mRNA was detected identically in both normal and alcoholic myocytes with the predicted size of 317 bp. For normalization, GAPDH was co-amplified with the predicted size of 500 bp. Compared with normal myocytes, alcoholic myocytes had a significantly higher signal ratio of β₃-AR mRNA to GAPDH, indicating increased β₃-AR gene expression in alcoholic monkeys.

Figure 7

Figure 7A LV myocyte β₁-AR protein expression following 12-month protocol. a) Examples of β₁-AR protein levels of myocytes obtained from two normal control and 2 alcoholic monkeys. One was a moderate drinker (M) and one was a heavy drinker (H). Actin was re-probed for normalization. b) Group means of β₁-AR protein levels of 3 groups of animals. Compared with normal control, myocyte β₁-AR protein levels were markedly decreased in both moderate and heavy drinkers. * P<0.05 vs normal; ** P<0.05 moderate vs heavy drinkers. Figure 7B LV myocyte β₃-AR protein expression following 12-month protocol. a) Examples of β₃-AR protein levels of myocytes obtained from two normal control and 2 alcoholic monkeys. One was a moderate drinker (M) and one was a heavy drinker (H). Actin was re-probed for normalization. b) Group means of β₃-AR protein levels of 3 groups of animals. Compared with normal control, myocyte β₃-AR levels were markedly increased in both moderate and heavy drinkers. Of note, compared with moderate drinkers, heavy alcohol intake caused more marked increases in cardiac β₃-AR. * P<0.05 vs normal; ** P<0.05 moderate vs heavy drinkers.

Figure 7

Figure 7A LV myocyte β₁-AR protein expression following 12-month protocol. a) Examples of β₁-AR protein levels of myocytes obtained from two normal control and 2 alcoholic monkeys. One was a moderate drinker (M) and one was a heavy drinker (H). Actin was re-probed for normalization. b) Group means of β₁-AR protein levels of 3 groups of animals. Compared with normal control, myocyte β₁-AR protein levels were markedly decreased in both moderate and heavy drinkers. * P<0.05 vs normal; ** P<0.05 moderate vs heavy drinkers. Figure 7B LV myocyte β₃-AR protein expression following 12-month protocol. a) Examples of β₃-AR protein levels of myocytes obtained from two normal control and 2 alcoholic monkeys. One was a moderate drinker (M) and one was a heavy drinker (H). Actin was re-probed for normalization. b) Group means of β₃-AR protein levels of 3 groups of animals. Compared with normal control, myocyte β₃-AR levels were markedly increased in both moderate and heavy drinkers. Of note, compared with moderate drinkers, heavy alcohol intake caused more marked increases in cardiac β₃-AR. * P<0.05 vs normal; ** P<0.05 moderate vs heavy drinkers.