Evidence for Arrhythmogenic Effects of A2A-Adenosine Receptors.

Adenosine can be released from the heart and may stimulate four different cardiac adenosine receptors. A receptor subtype that couples to the generation of cyclic adenosine monophosphate (cAMP) is the A2A-adenosine receptor (A2A-AR). To better understand its role in cardiac function, we studied mechanical and electrophysiological effects in transgenic mice that overexpress the human A2A-AR in cardiomyocytes (A2A-TG). We used isolated preparations from the left atrium, the right atrium, isolated perfused hearts with surface electrocardiogram (ECG) recording, and surface body ECG recordings of living mice. The hypothesized arrhythmogenic effects of transgenicity per se and A2A-AR stimulation were studied. We noted an increase in the incidence of supraventricular and ventricular arrhythmias under these conditions in A2A-TG. Moreover, we noted that the A2A-AR agonist CGS 21680 exerted positive inotropic effect in isolated human electrically driven (1 Hz) right atrial trabeculae carneae. We conclude that A2A-ARs are functional not only in A2A-TG but also in isolated human atrial preparations. A2A-ARs in A2A-TG per se and their stimulation can lead to cardiac arrhythmias not only in isolated cardiac preparations from A2A-TG but also in living A2A-TG.

Phenotyping of Mice with Heart Specific Overexpression of A2A-Adenosine Receptors: Evidence for Cardioprotective Effects of A2A-Adenosine Receptors.

Background: Adenosine can be produced in the heart and acts on cardiac adenosine receptors. One of these receptors is the A2A-adenosine receptor (A2A-AR). Methods and Results: To better understand its role in cardiac function, we generated and characterized mice (A2A-TG) which overexpress the human A2A-AR in cardiomyocytes. In isolated atrial preparations from A2A-TG but not from WT, CGS 21680, an A2A-AR agonist, exerted positive inotropic and chronotropic effects. In ventricular preparations from A2A-TG but not WT, CGS 21680 increased the cAMP content and the phosphorylation state of phospholamban and of the inhibitory subunit of troponin in A2A-TG but not WT. Protein expression of phospholamban, SERCA, triadin, and junctin was unchanged in A2A-TG compared to WT. Protein expression of the α-subunit of the stimulatory G-protein was lower in A2A-TG than in WT but expression of the α-subunit of the inhibitory G-protein was higher in A2A-TG than in WT. While basal hemodynamic parameters like left intraventricular pressure and echocardiographic parameters like the systolic diameter of the interventricular septum were higher in A2A-TG than in WT, after ß-adrenergic stimulation these differences disappeared. Interestingly, A2A-TG hearts sustained global ischemia better than WT. Conclusion: We have successfully generated transgenic mice with cardiospecific overexpression of a functional A2A-AR. This receptor is able to increase cardiac function per se and after receptor stimulation. It is speculated that this receptor may be useful to sustain contractility in failing human hearts and upon ischemia and reperfusion.

Cardiac function is regulated by B56α-mediated targeting of protein phosphatase 2A (PP2A) to contractile relevant substrates.

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of ß-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of -5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser(16) was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca(2+) sensitivity and increased basal contractility in TG hearts. These effects were reversed by ß-adrenergic stimulation.

Rapid enzyme-linked immunosorbent assay for the detection of antibodies against human neutrophil antigens -1a, -1b, and -1c.

BACKGROUND: Several methods exist for the detection of neutrophil antibodies; most of them, however, require fresh neutrophils. In this study, an enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-1 antigens (rHNAs) was developed to detect HNA-1a, -1b, and -1c alloantibodies in serum samples. STUDY DESIGN AND METHODS: Soluble rHNA-1a, -1b, and -1c were isolated from culture supernatant of transfected insect cells. Purified rHNA antigens were immobilized on microtiter wells using antibody against V5-Tag protein. Sera were added, and bound antibodies were detected by enzyme-labeled secondary antibodies. In parallel, monoclonal antibody-immobilized granulocyte antigen (MAIGA) was performed with two different monoclonal antibodies (MoAbs) against FcγRIIIb (3G8 and BW209). RESULTS: Fifteen MAIGA-positive sera containing HNA-1a alloantibodies were tested in ELISA. Thirteen of 15 (86.7%) MAIGA-positive sera captured by MoAbs 3G8 and/or BW209 reacted specifically with rHNA-1a. Four (26.7%) HNA-1a sera showed additional reaction with rHNA-1c. When anti-HNA-1b alloantibodies were analyzed in ELISA, 13 of 15 (86.7%) showed specific positive reaction with rHNA-1b, and 12 of 15 (80.0%) cross-reacted with rHNA-1c. Two HNA-1c sera reacted specifically with rHNA-1c. Immunoprecipitation analysis of all ELISA-negative HNA-1a and -1b sera did not show any specific band indicating false-positive reaction of these sera in MAIGA assay. CONCLUSIONS: These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA-1a, -1b, and -1c in patients with neutropenia and in blood donors.