Low- and high-level transgenic expression of beta2-adrenergic receptors differentially affect cardiac hypertrophy and function in Galphaq-overexpressing mice

Abstract

Transgenic overexpression of Galphaq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed beta-adrenergic receptor (betaAR) function. The role of betaAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the betaAR kinase (betaARK), which acts to inhibit the kinase, or concomitant overexpression of the beta2AR at low (approximately 30-fold, Galphaq/beta2ARL), moderate (approximately 140-fold, Galphaq/beta2ARM), and high (approximately 1,000-fold, Galphaq/beta2ARH) levels above background betaAR density. Expression of the betaARK inhibitor had no effect on the phenotype, consistent with the lack of increased betaARK levels in Galphaq mice. In marked contrast, Galphaq/beta2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and alpha-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized beta2AR/AC signal cannot be excluded. Higher expression of receptors in Galphaq/beta2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With approximately 1,000-fold overexpression, the majority of Galphaq/beta2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of betaAR signaling is deleterious in heart failure, selective enhancement by overexpressing the beta2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.

Figure 1

GRK activity in transgenic mice overexpressing Gαq in the heart. Activities of cytosolic preparations were determined in an in vitro assay with rod outer segments as substrate (see Materials and Methods). No differences were found between Gαq mice and nontransgenic littermates. Shown are the results from three independent experiments.

Figure 2

Effects of 30-fold β₂AR overexpression on left ventricular function in Gαq overexpressing transgenic mice. (A) Echocardiographic left ventricular shortening is depressed in Gαq overexpressors compared with nontransgenic controls (NTG). In Gαq/β₂AR_L mice, fractional shortening is normalized (n = 6–11). (B) Left ventricular +dP/dt_max at intrinsic heart rates (solid bars) and matched atrial paced heart rates (450 beats per min) (hatched bars) is depressed in Gαq overexpressors and normalized in the Gαq/β₂AR_L mice under both conditions (n = 3–5). ∗, P < 0.02 vs. NTG.

Figure 3

βAR signaling to AC in cardiac membranes from nontransgenic, Gαq-transgenic, and dual-transgenic Gαq/β₂AR_L mice. Basal (nonagonist) and maximal isoproterenol-stimulated activities were decreased in the Gαq mice (P < 0.02). ≈3-fold overexpression of β₂AR in the Gαq background (Gαq/β₂AR_L) had no effect on this signaling. Shown are results (mean ± SEM) from experiments performed with four mice from each group.

Figure 4

Effects of 140-fold β₂AR overexpression on ventricular function in Gαq-overexpressing transgenic mice. As shown, isoproterenol-stimulated increases in left ventricular fractional shortening were not observed in Gαq/β₂AR_M and Gαq mice. (n = 6 each); ∗, P < 0.05 vs. untreated; ^†, P < 0.05 vs. nontransgenic.

Figure 5

βAR signaling to AC in cardiac membrane from nontransgenic, Gαq-transgenic, and dual-transgenic Gαq/β₂AR_M mice. Overexpression of β₂AR to ≈140-fold in the Gαq mice resulted in enhanced basal and isoproterenol stimulated activities. Maximal activities were not different than NTG (P = 0.61), while the basal activities trended toward being lower, but not statistically different (P = 0.08), than NTG. Shown are mean results from experiments performed with four mice from each group.

Figure 6

Effects of 30- and 140-fold β₂AR overexpression on cardiac hypertrophy in Gαq-overexpressing transgenic mice. All mice were studied at 12–14 weeks of age. (A) Normalization of cardiac mass in Gαq overexpressors expressing β₂AR at lower levels (Gαq/β₂AR_L), but enhanced hypertrophy in Gαq with higher level β₂AR expression (Gαq/β₂AR_M) (n = 6–12). ∗, P < 0.05 vs. NTG. (B) Attenuation of hypertrophy-associated gene expression in hearts of Gαq/β₂AR_L, but not Gαq/β₂AR_M, mice. Sk act, α-skeletal actin. n = 6 per group. ∗, P < 0.01 vs. Gαq.

Figure 7

Myocardial fibrosis in Gαq/β₂AR_M transgenic mice. Masson’s trichrome stain of myocardial section from mid-left ventricular free wall of 8-week-old NTG (A), Gαq (B), Gαq/β₂AR_L (C), and Gαq/β₂AR_M (D). The perivascular blue staining serves as a control for the stain in that it identifies vascular collagen. Gαq/β₂AR_M exhibits significant fibrosis not observed in other groups (representative of 4–6 individual hearts examined). (Magnification, ×200.)