Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery

Abstract

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Figure 1

Expression of β-galactosidase in rabbit hearts after intracoronary in vivo delivery of a recombinant adenovirus. (a) X-Gal staining of a cross-section of a heart taken at midventricular level 6 days after intracoronary delivery of 1 × 10¹² tvp of Adeno-βGal. ×3. (b) Representative higher magnification of Adeno-βGal–treated hearts showing myocyte-specific β-Gal expression in cross (left) and longitudinal (right) sections. ×40. (c) Representative sections from hearts injected with EV (left) or Adeno-β₂AR (right). ×40.

Figure 2

β-AR overexpression in rabbit hearts after intracoronary delivery of Adeno-β₂AR. (a) Total β-AR density in different chambers of rabbit myocardium 6 days after 1 × 10¹² tvp of Adeno-β₂AR was delivered via intracoronary perfusion. β-AR density after Adeno-β₂AR treatment is compared with rabbit hearts that received the same dose of the EV. (b) Representative immunohistochemical detection of expressed human β₂-ARs in rabbit LV 6 days after intracoronary delivery of Adeno-β₂AR. (c) Dose-dependent myocardial β₂-AR overexpression after delivery of increasing doses of Adeno-β₂AR. Total β-AR density is shown in the RV and LV compared with myocardial β-AR density after delivery of EV, which did not change from endogenous β-AR levels. (d) Time course of β-AR overexpression in rabbit hearts after delivery of 5 × 10¹¹ tvp Adeno-β₂AR. All binding values represent the mean ± SEM.

Figure 3

Membrane adenylyl cyclase activity from hearts of Adeno-β₂AR–treated rabbits. Membranes were purified from rabbits 6 days after intracoronary delivery of 5 × 10¹¹ tvp of Adeno-β₂AR or EV (n = 6 each), and adenylyl cyclase activity was measured under basal conditions and after isoproterenol (10^–4 M) or NaF (10 mM). Values shown are the mean ± SEM normalized to the value seen with NaF. Activity in the 2 groups with NaF was not significantly different (data not shown), indicating no postreceptor defects in signaling. *P < 0.05 vs. EV (Student’s t test).

Figure 4

In vivo assessment of LV contractile function in rabbits treated with Adeno-β₂AR. Cardiac catheterization was performed in conscious sedated rabbits 6 days after intracoronary delivery (1.5 mL in PBS) of either 5 × 10¹¹ tvp Adeno-β₂AR (n = 11), EV (n = 7), or saline (n = 11) using a high-fidelity micromanometer. Parameters measured include (a) LV +dP/dt_max and (b) LVSP. Shown are the baseline values (as mean ± SEM) and responses to progressive doses of isoproterenol. *P < 0.05; ^#P < 0.005 vs. saline (Student’s t test). For LV +dP/dt_max and LVSP, isoproterenol responses in the Adeno-β₂AR group are significantly different than those in the EV-treated group (P < 0.01, ANOVA).

Figure 5

Relationship between myocardial β₂-AR overexpression and in vivo contractility. Total cardiac β-AR density was plotted against maximal isoproterenol-stimulated LV +dP/dt_max of rabbits treated with Adeno-β₂AR. A significant linear relationship was found (t test).

Figure 6

In vivo assessment of LV contractile function (LV +dP/dt_max) in rabbits 21 days after treatment with Adeno-β₂AR. Cardiac catheterization was performed in conscious sedated rabbits after intracoronary delivery (1.5 mL in PBS) of either 5 × 10¹¹ tvp Adeno-β₂AR (n = 9) or EV (n = 5) using a high-fidelity micromanometer. Shown are the baseline values (mean ± SEM) and responses to progressive doses of isoproterenol. *P < 0.05 vs. EV (Student’s t test). Total β-AR density after Adeno-β₂AR treatment was 115 ± 12 fmol/mg membrane protein vs. 79 ± 7 in hearts that received the same dose of EV (P < 0.05, Student’s t test).

Figure 7

Rescue of attenuated signaling in failing ventricular myocytes by treatment with Adeno-β₂AR. Shown is the isoproterenol-induced cAMP production in sham and failing isolated ventricular cardiomyocytes treated with EV or Adeno-β₂AR. Failing ventricular myocytes were isolated from rabbit hearts 21 days after left circumflex coronary artery ligation (21). Experiments were performed 36 hours after adenoviral infection and after overnight labeling with 1.5 μCi/mL [³H]adenine (see Methods). The accumulation of intracellular cAMP is expressed as percent conversion from total ³H uptake. The data represent the mean ± SEM of 4–9 independent experiments, each performed in triplicate. Total responses of the failing/EV group were significantly attenuated compared with sham/EV-treated myocytes (P < 0.05, single-factor ANOVA). Total responses of the failing Adeno-β₂AR–treated myocytes were significantly enhanced compared with the failing/EV-treated cells (*P < 0.05, ANOVA).