Gene dosage-dependent effects of cardiac-specific overexpression of the A3 adenosine receptor

Abstract

We used a genetic approach to determine whether increasing the level of A3 adenosine receptors (A3ARs) expressed in the heart confers protection against ischemia without causing cardiac pathology. We generated mice carrying one (A3tg.1) or six (A3tg.6) copies of a transgene consisting of the cardiomyocyte-specific alpha-myosin heavy chain gene promoter and the A3AR cDNA. A3tg.1 and A3tg.6 mice expressed 12.7+/-3.15 and 66.3+/-9.4 fmol/mg of the high-affinity G protein-coupled form of the A3AR in the myocardium, respectively. Extensive morphological, histological, and functional analyses demonstrated that there were no apparent abnormalities in A3tg.1 transgenic mice compared with nontransgenic mice. In contrast, A3tg.6 mice exhibited dilated hearts, expression of markers of hypertrophy, bradycardia, hypotension, and systolic dysfunction. When A3tg mice were subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion, infarct size was reduced approximately 30% in A3tg.1 mice and approximately 40% in A3tg.6 mice compared with nontransgenic littermates. The reduction in infarct size in the transgenic mice was not related to differences in risk region size, systemic hemodynamics, or body temperature, indicating that the cardioprotection was a result of increased A3AR signaling in the ischemic myocardium. The results demonstrate that low-level expression of A3ARs in the heart provides effective protection against ischemic injury without detectable adverse effects, whereas higher levels of A3AR expression lead to the development of a dilated cardiomyopathy.

Figure 1

A, Diagram of the transgene construct used for the generation of A₃AR transgenic mice. Construct contains the α-MyHC gene promoter, the full-length canine A₃AR cDNA clone, and a human growth hormone (Hgh) polyadenylylation sequence. Solid line indicates the probe used for Southern analysis; dashed line, region amplified by PCR for genotyping. B, Representative Southern blot of genomic DNA from A₃tg.1 (lane 1, nontransgenic; lane 2, transgenic) and A₃tg.6 (lane 3, non-transgenic; lane 4, transgenic) mice. DNA (30 μg) was digested with EcoRI and subsequently probed with a cDNA probe corresponding to the α-MyHC gene promoter. Transgene copy number was determined by comparing the intensity of the endogenous gene (2.6-kb fragment) and the transgene (3.6-kb fragment). C, Northern blot analysis of total RNA (10 μg/lane) isolated from the hearts of A₃tg.1 mice (lane 1, nontransgenic; lane 2, transgenic) and A₃tg.6 (lane 3, nontransgenic; lane 4, transgenic) mice. Blots were probed with a full-length canine A₃AR cDNA probe.

Figure 2

Quantification of A₃AR expression in transgenic mouse hearts by radioligand binding analysis. A, Demonstration of specific binding of ¹²⁵I-AB-MECA to heart membranes prepared from nontransgenic (NTG), A₃tg.1, and A₃tg.6 mice. Each reaction contained 100 μg of membrane protein, ≈0.3 nmol/L ¹²⁵I-AB-MECA (100 000 cpm), and 300 nmol/L WRC 0571 to block binding to A₁ARs. Values are the mean±SEM (n=3). *P<0.05 vs the vehicle group by Student's t test. B, Equilibrium binding analysis with membranes prepared from A₃tg.1 and A₃tg.6 hearts. Scatchard transformation of the data (inset) demonstrates that ¹²⁵I-AB-MECA binds to 2 affinity states of the A₃AR. Affinities and binding capacities for the 2 binding sites for each line are reported in Table 1. Each reaction contained 100 μg membrane protein and 300 nmol/L WRC 0571. Data are representative of 3 independent experiments.

Figure 3

Representative hematoxylin and eosin stain of left ventricular sections (100×) from nontransgenic (NTG), A₃tg.1, and A₃tg.6 transgenic mouse hearts. Myocardial disarray was evident in A₃tg.6 hearts.

Figure 4

A, Representative M-mode echocardiograms of a nontransgenic (NTG) mouse, an A₃tg.1 transgenic mouse, and an A₃tg.6 mouse. B, Representative dot blots of ANF, α-skel actin, α-MyHC, and β-MyHC of total heart RNA (3 μg) from NTG, A₃tg.1, and A₃tg.6 mice at increasing ages. Blots include duplicate samples of RNA from 2 separate mice at each time point. Equal loading of the samples was verified by reprobing the blots with GAPDH.

Figure 5

Myocardial infarct size after in vivo coronary occlusion/reperfusion. A, Myocardial infarct size expressed as a percentage of the risk region. Filled circles indicate the mean±SEM; open circles, individual mice. *P<0.05 vs NTG mice by 1-way ANOVA followed by Student's t test and the Bonferroni correction. B, Representative images of heart slices obtained after staining with phthalo blue dye and TTC used to assess infarct size.