The generation of β₁AR and β₂AR TG mice that overexpress human cardiac-specific β₁- or β₂ARs was performed as previously described (6,7). In brief, wild-type human β₁- and β₂-AR cDNA was ligated into the SalI site (exon 3) of the full-length 5.5-kb α-myosin heavy chain (MHC) promoter, obtained from Dr. Arthur R. Struch, (Mayo Clinic, Rochester, MN, USA). The linearized constructs were injected into male pronuclei of fertilized FVB/N mouse oocytes and implanted into pseudopregnant female oviducts (Taconic Biosciences, Rensselaer, NY, USA). Genomic DNA from tail-cuts was screened for the presence of transgenes, using targeted PCR with the following primers: For β₁AR, forward primer 5′-AGGACTTCACATAGAAGCCTAG-3′, located in the α-MHC promoter, and reverse primer 5′-TGTCCACTGCTGAGACAGCG-3′, located in the β₁AR coding sequence. For β₂AR, forward primer 5′-GGAGCAGAGTGGATATCACG-3′, located in the open reading frame, and reverse primer 5′-GTCACACCACAGAAGTAAGG-3′, located in the SV40 polyadenylation region. A total of 39 male mice (n=13 mice/group) were housed in individual cages (temperature, 23°C; humidity, 60%) under 12/12 h light/dark cycles, and provided with commercial chow and water ad libitum. Mice were examined at 6 months of age, to ensure the presented phenotypes were independent of the confounding effects of cardiac growth and associated alterations to βAR functional coupling. Animal procedures were approved by the University of Maryland Baltimore Institutional Animal Care and Use Committee.

Physiological parameters were evaluated in an isolated work-performing experiment, according to a previously published procedure (7). Briefly, mice (n=5/group; age, 6 months; weight, ~35 g) were anesthetized via intraperitoneal injection of 100 mg/kg Nembutal and 1.5 units of heparin to prevent microthrombosis, the heart and aorta were attached to a 20-gauge cannula and subjected to temporary retrograde perfusion with oxygenated Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl_2, 0.5 mM Na-EDTA, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 11 mM glucose; followed by saturation with 95% O₂+5% CO₂). A polyethelene-50 catheter was inserted into the apex of the left ventricle to measure intraventricular pressure. The pulmonary vein was connected to a second cannula and the antegrade perfusion was initiated with a basal workload of 300 mmHg ml/min (venous return, 6 ml; mean aortic pressure, 50 mmHg). Hearts were allowed to equilibrate for 20 min. Atrial pressure was monitored through a sidearm in the left atrial cannula (National Instruments Corporation, Austin, Texas, US). The left ventricular pressure signals were analyzed off-line using NI Biobench software version 1.0 (National Instruments Corporation). The first positive and negative derivatives of the left intraventricular pressure curve [that is, the maximal rate pressure development (+dP/dt) and the maximal rate pressure decline (−dP/dt)], the duration of contraction and relaxation [that is, time to peak pressure (TPP)], and the time to half relaxation (TR½) were calculated. TPP was normalized with respect to peak systolic pressure, calculated as: Systolic pressure-end diastolic pressure. TR½ was normalized with respect to ½ relaxation pressure, calculated as: (Systolic pressure-diastolic pressure)/2.

Mice (n=5/group) were anesthetized via intraperitoneal injection of pentobarbital sodium (100 mg/kg). Hearts were isolated and fixed with 10% buffered formalin for 2 days at room temperature. The heart/body weight ratio was calculated according to the following formula: Heart/body weight ratio=heart weight (mg)/body weight (g). Hearts were embedded in paraffin and sectioned (6 µm). The sections were stained with H&E, and digital images were captured using a light microscope and analyzed using the digital imaging analysis software Leica Steel Expert version 2.0 (Leica Microsystems GmbH, Wetzlar, Germany). Cardiomyocyte diameter was determined as the shortest distance across the nucleus in transverse cell sections. Cardiomyocytes (n=100) from 5 randomly selected microscope fields (x200 magnification) from the posterior wall of the left ventricle were measured to represent the average cardiomyocyte diameter.

The expression of heart proteins was detected according to previously published procedures (15). Briefly, proteins were extracted from heart tissue (~50 mg tissue; n=3 mice/group) using radioimmunoprecipitation assay buffer (1:4 w:v; R2002, Biosesang, Inc., Soungnam, Korea) containing 150 mM NaCl, 1% Triton X-100, 1% sodium deooxycholate, 50 mM Tris HCl (pH 7.5), 0.1% SDS, 2 mM EDTA (pH 8.0) and phosphatase inhibitor cocktail for 10 min over ice. Lysates were centrifuged at 19,000 × g for 10 min at 4°C and supernatants were collected. Protein concentrations were determined using a bicinchoninic acid assay kit. Equal amounts of extracted protein samples (200 mg/ml) were subjected to Tris-Glycine-SDS PAGE, transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat skim milk in TBS containing 0.1% Tween 20 for 2 h at room temperature and probed with the following primary antibodies overnight at 4°C: Anti-Gs (1:500; cat no. sc26766), anti-Gi2 (1:500; cat no. sc391), anti-Gi3 (1:500; cat no. sc262), anti-G-protein-coupled receptor kinase 2 (GRK2; 1:500; cat no. sc562), anti-GATA binding protein 4 (GATA4; 1:1,000; cat no. sc1237) and anti-GAPDH (1:2,000; cat no. sc166574), obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-VEGF-A (1:1,000; cat no. ab1316) and anti-phosphorylated (p)-GATA4 (1:500; cat no. ab5245) obtained from Abcam (Cambridge, MA, USA). Membranes were then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat no. SC-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. Protein bands were visualized using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences, Chalfont, UK) and the signals were acquired using a LAS-3000 charged coupled device camera (Fujifilm Holdings Corporation, Tokyo, Japan). Blots were semi-quantified using densitometric analysis and protein expression was normalized to GAPDH. Semi-quantification was performed using the Scion Image software version 4.0.3.2 (Scion Corporation, Walkersville, MD, USA). Experiments were performed in triplicate.

All data are presented as the mean ± standard error of the mean. Differences of the means amongst the groups were estimated by one-way analysis of variance followed by a post hoc Bonferroni test for multiple comparisons. All analyses were performed using SPSS version 20.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The cardiac function of β₁AR TG and β₂AR TG mice was enhanced, and the cardiac contractility/relaxation and heart rate of β₁- and β₂AR TG mice were significantly increased compared with the WT mice (Table I). No significant differences between the β₁AR and β₂AR TG mouse hearts were identified; however, the clinical parameters, including left ventricular systolic pressure (SP), left ventricular diastolic pressure (DP), left ventricular end-diastolic pressure (EDP) and TPP in β2AR TG mice appeared to be slightly increased compared with in β1AR TG mice.

Heart/body weight ratio was significantly greater in β₁AR and β₂AR TG mice compared with in WT mice (3.91±0.07 and 3.78±0.06 mg/g vs. 3.61±0.03 mg/g, respectively; P<0.05); however, no statistical significance was identified between the two transgenic groups. The diameter of the cardiomyocytes demonstrated a similar trend to the heart/body weight ratios; cell diameters were greater in βAR TG mice compared with in the WT mice, however, no significant differences in cardiomyocyte size were detected between the two transgenic groups (Fig. 1).

Protein expression levels of GRK2, Gs, Gi2 and Gi3 were examined by western blot analysis (Fig. 2). Gs protein expression level was significantly decreased, whereas Gi2, Gi3 and GRK2 expressions were significantly increased, in β₁AR and β₂AR TG mice compared with WT mice (Fig. 2). The upregulation of Gi2 was greatest in the β₂AR TG mice compared with the other two groups.

Upregulation of VEGF-A expression was detected in both β₁AR and β₂AR TG mice; however, the β₂AR TG mice exhibited a smaller increase compared with β₁AR TG mice. Furthermore, GATA4 serine 105 phosphorylation (p-GATA4) was similarly increased in both β₁ and β₂AR TG mice, compared with the WT group. Phosphorylation on serine 105 is an event known to augment GATA4 activity. No significant difference in pGATA4 expression was observed between the β₁AR and β₂AR TG mice (Fig. 3).