Myocardial β(2) -adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failure

Abstract

Background and purpose: We investigated whether β(2) -adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart.

Experimental approach: We explored the angiogenic effects of β(2) -adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated β(2) -adrenoceptor overexpression was obtained via direct intramyocardial injection 4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observation to β(2) -adrenoceptor -/- mice undergoing MI.

Key results: Transgenes were robustly expressed in the LV at 2 weeks post-gene therapy, whereas their expression was minimal at 4-weeks post-gene delivery. In HF rats, cardiac β(2) -adrenoceptor overexpression resulted in enhanced basal and isoprenaline-stimulated cardiac contractility at 2-weeks post-gene delivery. At 4 weeks post-gene transfer, Ad-β(2) -adrenoceptor HF rats showed improved LV remodeling and cardiac function. Importantly, β(2) -adrenoceptor overexpression was associated with a markedly increased capillary and arteriolar length density and enhanced in vivo myocardial blood flow and coronary reserve. At the molecular level, cardiac β(2) -adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway. In β(2) -adrenoceptor-/- mice, we found a ~25% reduction in cardiac capillary density compared with β(2) -adrenoceptor+/+ mice. The lack of β(2) -adrenoceptors was associated with a higher mortality rate at 30 days and LV dilatation, and a worse global cardiac contractility compared with controls.

Conclusions and implication: β(2) -Adrenoceptors play an important role in the regulation of the angiogenic response in HF. The activation of VEGF/PKB/eNOS pathway seems to be strongly involved in this mechanism.

Figure 1

(A)Overall design of the 8-week study. (B) Representative GFP fluorescence microscopy (left), light microscopy (middle) and overlay of both (right) of LV myocardium 2 weeks after intramyocardial Ad-GFP delivery. (C) β₂-Adrenoceptor (AR) immunohistochemistry in LV tissue from Ad-GFP- (left, control) and Ad-β₂-adrenoceptor-treated (right) rats (2 weeks post-gene delivery). Magnification ×40. (D) Total β-adrenoceptor density (B) in cardiac homogenates purified from hearts of HF Ad-β₂-adrenoceptor and HF control (HF-saline and HF Ad-GFP) groups at 2 weeks after gene therapy (n= 6 and 8 for each group); *P < 0.001 vs. HF control. (E) Right panel; percentage (%) of GFP-stained isolated myocytes assessed 2 and 4 weeks following Ad-GFP in vivo gene delivery to HF rats by direct intra-myocardial injection (n= 5 for each time point). Green myocytes from each rat heart were counted in five randomly selected fields and expressed as percentage of the total number of myocytes per field. *P < 0.001 vs. 4 weeks after gene delivery. Data are presented as means ± SEM. Left panel: representative GFP fluorescence microscopy (upper), light microscopy (middle) and overlay of both (lower) of myocytes 2 weeks after Ad-GFP delivery.

Figure 2

(A) Average LV +dP/dt and LV −dP/dt values (B) at 2 weeks post-gene therapy in the four experimental groups evaluated under basal conditions and after isoprenaline stimulation. (C) Average left ventricle end diastolic pressure (LVEDP) in the four experimental groups. Sham, n= 11; HF-saline, n= 13; HF Ad-GFP, n= 12; HF Ad- β₂-adrenoceptor (AR), n= 12. anova analysis and Bonferroni test were used among all groups. Data are presented as mean ± SEM. *P < 0.05 vs sham at basal or at each respective dose of isoprenaline; †P < 0.05 vs HF-saline and HF Ad-GFP at basal or at each respective dose of isoprenaline.

Figure 3

(A) Representative LV cross sections and (B) echocardiographic M-mode recordings from of all study groups at the end of the study period. (C) LV internal diameter at diastole (LVIDd) (left), LVID at systole (LVIDs) (middle) and fractional shortening (FS) (right) as measured by echocardiography 4 weeks after gene delivery. Sham, n= 11; HF-saline, n= 14; HF Ad-GFP, n= 11; HF Ad-β₂-adrenoceptor (AR), n= 12. Bar = 10 mm. anova analysis and Bonferroni test among all groups. All data are expressed as mean ± SEM. *P < 0.05 vs. sham at basal or at each respective dose of isoprenaline; †P < 0.05 vs. HF-saline and HF Ad-GFP at basal or at each respective dose of isoprenaline.

Figure 4

(A) Representative images of (left) Lectin Bandeiraea simplicifolia I staining of capillaries in LV sections and (right) of arterioles stained with antibodies against smooth muscle α-actin obtained from all study groups at 4 weeks post-gene therapy in the lateral wall far from the infarcted area (remote). Magnification ×40. Scale bar: 50 µm. (B) Histograms show data on capillary counts, and (C) arteriolar length density in either LV border anterior and lateral, and remote zones in all study groups at 4 weeks after gene therapy (n= 5 for each group). (D) Average of myocardial blood flow at basal condition and after maximal coronary dilation by dipyridamole and of coronary reserve measured in all study groups at the end of the study period (n= 8 rats for each group). anova analysis and Bonferroni test among all groups. All data are expressed as mean ± SEM. *P < 0.05 vs. sham; †P < 0.05 vs. HF-saline and HF Ad-GFP.

Figure 5

Shown is cardiac protein expression of VEGF, PKB, Ser⁴⁷³-phospho(p)-PKB, eNOS and Ser¹¹⁷⁷-phospho(p)-eNOS in all study groups at 2 (A) and 4 (B) weeks after gene therapy. Data between HF-saline and Ad-GFP were not statistically different and were pooled together and indicated as HF-control. The expression of GAPDH was used as an internal control to normalize VEGF protein levels. p-PKB/PKB and p-eNOS/eNOS ratio indicated respectively the levels of PKB and eNOS phosphorylation in the heart. Data are expressed as mean ± SEM. *P < 0.05 vs. sham (n= 6 rats per each group, for each time point).

Figure 6

(A) Post-MI 30-day mortality rate in HF β₂-adrenoceptor (AR)+/+ and β₂-adrenoceptor−/− mice (n= 35 mice for each group). (B) LV fractional shortening FS (%) assessed by echocardiography at 4 weeks post-MI or sham operation in β₂-adrenoceptor+/+ and β₂-adrenoceptor−/− mice (n= 15 for each group). (C) Capillary counts (expressed as total capillary density µm^-2) in LV remote zone in all study groups (n= 8 for each group). (D) Shown is the cardiac protein expression of Ser⁴⁷³p-PKB/PKB (left), Ser¹¹⁷⁷-p-eNOS/eNOS and (middle) and VEGF (right) in sham or HF β₂-adrenoceptor+/+ and β₂-adrenoceptor−/− at the end of the study (n= 8 for each group). Data are expressed as mean ± SEM. *P < 0.05 vs. sham β₂-adrenoceptor+/+; †P < 0.05 vs. HF β₂-adrenoceptor+/+.