Cardioprotection of controlled and cardiac-specific over-expression of A(2A)-adenosine receptor in the pressure overload

Abstract

Adenosine binds to three G protein-coupled receptors (R) located on the cardiomyocyte (A(1)-R, A(2A)-R and A(3)-R) and provides cardiac protection during both ischemic and load-induced stress. While the role of adenosine receptor-subtypes has been well defined in the setting of ischemia-reperfusion, far less is known regarding their roles in protecting the heart during other forms of cardiac stress. Because of its ability to increase cardiac contractility and heart rate, we hypothesized that enhanced signaling through A(2A)-R would protect the heart during the stress of transverse aortic constriction (TAC). Using a cardiac-specific and inducible promoter, we selectively over-expressed A(2A)-R in FVB mice. Echocardiograms were obtained at baseline, 2, 4, 8, 12, 14 weeks and hearts were harvested at 14 weeks, when WT mice developed a significant decrease in cardiac function, an increase in end systolic and diastolic dimensions, a higher heart weight to body weight ratio (HW/BW), and marked fibrosis when compared with sham-operated WT. More importantly, these changes were significantly attenuated by over expression of the A(2A)-R. Furthermore, WT mice also demonstrated marked increases in the hypertrophic genes β-myosin heavy chain (β-MHC), and atrial natriuretic factor (ANF)--changes that are mediated by activation of the transcription factor GATA-4. Levels of the mRNAs encoding β-MHC, ANP, and GATA-4 were significantly lower in myocardium from A(2A)-R TG mice after TAC when compared with WT and sham-operated controls. In addition, three inflammatory factors genes encoding cysteine dioxygenase, complement component 3, and serine peptidase inhibitor, member 3N, were enhanced in WT TAC mice, but their expression was suppressed in A(2A)-R TG mice. A(2A)-R over-expression is protective against pressure-induced heart failure secondary to TAC. These cardioprotective effects are associated with attenuation of GATA-4 expression and inflammatory factors. The A(2A)-R may provide a novel new target for pharmacologic therapy in patients with cardiovascular disease.

Figure 1. Over-expression of the A_2A adenosine receptor in mice myocardium.

Mice with constitutive and controlled overexpression of A_2A-R were created. (A & B) Bi-transgenic, cardiac specific doxycyline regulated A_2A-R transgenic mice were generated and confirmed there is A_2A-R expression in all of lines; (C & D) A representative diagram of the timeline of gene induction. The constitutive model was not placed on doxycyline and over expressed A_2A-R at birth while the controlled or induced model was placed on doxcycline during mating and removed at the age of 3 weeks.

Figure 2. Effects of cardiac specific A_2A-R e

xpression on cardiac funtion and calcium handeling. (A) Echocardiography of mice with inducible, cardiac restricted expression of A_2A-R TG and wild type (WT) mice. Fraction shorting (FS) at 8 week and 24 weeks in A_2A-R TG and WT mice showed persist hyper-contractile phenotype in A_2A-R TG mice up to 24 weeks (*p<0.01 vs WT mice, n = 8). (B & C) Calcium transient data showing increased systolic calcium (B) and rapid calcium re-uptake activity (C) in cardiomyocytes from the A_2A-R TG mice at 10 weeks age compared to WT. Data were expressed as mean±SE. *p<0.01 compared to WT cardiomyocytes, n = 15 cells from 5 mice hearts.

Figure 3. Effect of TAC on left ventricular hypertrophy and function as measured by Echocardiography.

(A) A_2A-R mice had preserved cardiac function and showed tolerance to TAC-induced pressure overload. WT mice developed a significant decrease in cardiac contractile function at 14 weeks after TAC (*P<0.01, repeated measures ANOVA test, n = 17). Of note, contractile function in A_2A-R TG mice was slightly decline after TAC. *p<0.01 vs WT TAC at the same time point. (B &C) WT mice developed into a significant increase in end systolic dimensions (B) and a higher heart weight to body weight ratio (C).*p<0.01 vs sham group, n = 8–17; ^#p<0.01 vs WT TAC, n = 10–17. (D) WT mice showed significantly more fibrosis than A_2A-R TG at 14 weeks post TAC. Data was expressed as mean±SE *p<0.01 vs WT, n = 10–17. The basal fibrosis is no difference between WT and A_2A-R TG. Both were below 0.1% of total myocardium area.

Figure 4. Effect of TAC on GATA-4, ANP, β-MHC and A1-R expression in A_2A-R and WT mice.

After 14 weeks of TAC, the total RNA was isolated from either A_2A-R TG or WT mice myocardium. The Q-PCR was performed to check the gene expression of GATA-4 ANP, β-MHC and A₁-R. Data were expressed as mean ± SE. ^∗p<0.05 vs sham, ^#p<0.05 vs WT mice TAC group, n = 6.

Figure 5. Effects of TAC on Inflammatory genes expression in A_2A and WT Mice.

After 14wks of TAC, the total RNA was isolated from A_2A-R TG and WT mice myocardium. The Q-PCR was performed to check the gene expression of cysteine dioxygenase 1 (Cdo1), complement component 3 (C3), serine (or cysteine) peptidase inhibitor, member 3N (Serpina3n), and toll-like receptor (TLr 7). Data was expressed as mean ± SE. *p<0.05 vs sham, ^#p<0.05 vs WT mice TAC group, n = 6. ^&p<0.05 vs WT sham.