Cardiac-restricted overexpression of the A(2A)-adenosine receptor in FVB mice transiently increases contractile performance and rescues the heart failure phenotype in mice overexpressing the A(1)-adenosine receptor

Abstract

In the heart, adenosine binds to pharmacologically distinct G-protein-coupled receptors (A(1)-R, A(2A)-R, and A(3)-R). While the role of A(1)- and A(3)-Rs in the heart has been clarified, the effect of genetically manipulating the A(2A)-R has not been defined. Thus, we created mice overexpressing a cardiac-restricted A(2A)-R transgene. Mice with both low (Lo) and high (Hi) levels of A(2A)-R overexpression demonstrated an increase in cardiac contractility at 12 weeks. These changes were associated with a significantly higher systolic but not diastolic [Ca(2+)]i, higher maximal contraction amplitudes, and a significantly enhanced sarcoplasmic reticulum Ca(2+) uptake activity. At 20 weeks, the effects of A(2A)-R overexpression on cardiac contractility diminished. The positive effects elicited by A(2A)-R overexpression differ from the heart failure phenotype we observed with A(1)-R overexpression. Interestingly, coexpression of A(2A)-R TG(Hi), but not A(2A)-R TGLo, enhanced survival, prevented the development of left ventricular dysfunction and heart failure, and improved Ca(2+) handling in mice overexpressing the A(1)-R. These results suggest that adenosine-mediated signaling in the heart requires a balance between A(1)- and A(2A)-Rs--a finding that may have important implications for the ongoing clinical evaluation of adenosine receptor subtype-specific agonists and antagonists for the treatment of cardiovascular diseases.

Keywords: Ca2+ transients; adenosine receptors; cardiac myocytes; heart failure; transgenic mice.

Figure 1

Creation of transgenic mice expressing cardiac‐specific a2a‐r. (a) 
A_2a‐R transgenic founder lines expressing low and high levels of A_2a‐R. Total ventricular protein extracts from 6‐week‐old male mice were immunoblotted with anti‐A_2a‐R antibody.
(B) Immunofuorescence staining of isolated myocytes showing A_2a‐R and α‐actinin. 60× whole cell and enlarged images are shown.
(C) Morphology of isolated myocytes and their length and width measurements. 20× images are shown. Values are means +/‐SE. n= 38–56 myocytes pooled from four to five mice.

Figure 2

Genomic copies of inserted A_2a‐R transgene. Copy number was calculated by real‐time pcr using 100 ng of genomic dna and mouse/human specifc A_2a‐R primers. Data were normalized to actin and wild‐type A_2a‐R gene values. Each sample was measured in triplicate and 4–6 samples were used for each genotype. Graph is shown with standard error.

Figure 3

Creation of transgenic mice overexpressing both A₁‐R and A_2A‐R. (A) A₁‐R and A_2A‐R expression in WT, A1‐TG, A_2A‐TG and A1/A_2A‐TG mice. Ventricular extracts from 8‐ week‐old male mice were probed with indicated antibodies. (B) A₁‐R and A_2A‐R colocalized on cardiac myocyte membrane. 60× confocal images are shown. (C) Horizontal sections of mouse hearts stained with hematoxylin‐eosin (HE). Minimum two animals (8‐week‐old male) of each genotype were stained and representative scaled photographic images (1×) are shown.

Figure 4

A_2a‐R expression improved cardiac function and hemodynamics without affecting heart rate in A₁‐TG mice. (A) Percent fractional shortening of indicated mouse groups; (B) Heart rate of indicated mouse groups; (C) and (D) Hemodynamic functions of transgenic mice: maximum rate of ventricular pressure rise and decline (+ DP/DT and ‐DP/DT.) *p < 0.001 versus WT, †p < 0.001 versus A₁TG. (n= 15–21, SE, male 8–12‐week‐old mice).

Figure 5

Animal survival, calcium handling, and gene expression in A₁TG and A_2a‐TG mice. (a) Kaplan‐Meier survival curve for A₁‐R transgenic lines coexpressing high levels of A_2a‐R (A_2a‐TGHi). A1 versus A₁/A_2a p < 0.001. (B) Kaplan‐Meier survival curve for A₁‐R transgenic lines coexpressing low levels of A_2a‐R (A_2a‐TGlo). A₁ versus A₁/A_2a NS. (C) Representative tracings of myocyte Ca²⁺ transients and contractions. Detailed calculations are shown in Table 2. (D) SERCA2 and Gαi2 expression. Ventricular extracts from 8‐week‐old male mice were probed with indicated antibodies. SERCA2 and Gαi2 signals were normalized to average intensity in WT hearts (n= 4–7) and were analyzed by one‐way analysis of variance followed by Dunnett's test. Analyzed values were mean +/‐ SE, *p < 0.05 versus WT.

Figure 6

Pico Sirius Red staining of 12‐week and 20‐week‐old A_2a‐TG_hi myocardium (collagen‐stained purple). Representative 10× objective images are shown. Two independent observers evaluated fbrosis based on 4–7 samples/group.