Role of transglutaminase 2 in A1 adenosine receptor- and β2-adrenoceptor-mediated pharmacological pre- and post-conditioning against hypoxia-reoxygenation-induced cell death in H9c2 cells

Abstract

Pharmacologically-induced pre- and post-conditioning represent attractive therapeutic strategies to reduce ischaemia/reperfusion injury during cardiac surgery and following myocardial infarction. We have previously reported that transglutaminase 2 (TG2) activity is modulated by the A₁ adenosine receptor and β₂-adrenoceptor in H9c2 cardiomyoblasts. The primary aim of this study was to determine the role of TG2 in A₁ adenosine receptor and β₂-adrenoceptor-induced pharmacological pre- and post-conditioning in the H9c2 cells. H9c2 cells were exposed to 8h hypoxia (1% O₂) followed by 18h reoxygenation, after which cell viability was assessed by monitoring mitochondrial reduction of MTT, lactate dehydrogenase release and caspase-3 activation. N⁶-cyclopentyladenosine (CPA; A₁ adenosine receptor agonist), formoterol (β₂-adrenoceptor agonist) or isoprenaline (non-selective β-adrenoceptor agonist) were added before hypoxia/reoxygenation (pre-conditioning) or at the start of reoxygenation following hypoxia (post-conditioning). Pharmacological pre- and post-conditioning with CPA and isoprenaline significantly reduced hypoxia/reoxygenation-induced cell death. In contrast, formoterol did not elicit protection. Pre-treatment with pertussis toxin (G_i/o-protein inhibitor), DPCPX (A₁ adenosine receptor antagonist) or TG2 inhibitors (Z-DON and R283) attenuated the A₁ adenosine receptor-induced pharmacological pre- and post-conditioning. Similarly, pertussis toxin, ICI 118,551 (β₂-adrenoceptor antagonist) or TG2 inhibition attenuated the isoprenaline-induced cell survival. Knockdown of TG2 using small interfering RNA (siRNA) attenuated CPA and isoprenaline-induced pharmacological pre- and post-conditioning. Finally, proteomic analysis following isoprenaline treatment identified known (e.g. protein S100-A6) and novel (e.g. adenine phosphoribosyltransferase) protein substrates for TG2. These results have shown that A₁ adenosine receptor and β₂-adrenoceptor-induced protection against simulated hypoxia/reoxygenation occurs in a TG2 and G_i/o-protein dependent manner in H9c2 cardiomyoblasts.

Keywords: A(1) adenosine receptor; Cardiomyocytes; Post-conditioning; Pre-conditioning; Transglutaminase 2; β(2)-adrenoceptor.