α_1A-adrenoceptor stimulation promotes glucose uptake and cell survival in cardiomyocytes - role of mTOR

Abstract

Background: There has been convincing evidence that cardiac α 1A -adrenoceptors (α 1A -ARs) increase in expression and have an enhanced compensatory role during advanced heart failure (O'Connell et al, 2014). Mechanistic target of rapamycin (mTOR) signalling is involved in many critical cellular processes, and implicated in early development, normal function and stress responses of cardiomyocytes. However, it remains unclear whether mTOR is involved in the signalling mechanisms underlying α 1A -AR-mediated cardioprotective effects. This study aims to elucidate the ability of α 1A -ARs to enhance cardiomyocyte viability via mTOR signalling. Methods: Neonatal rat ventricular myocytes (NRVMs) were incubated under normoxic or hypoxic (1% O 2 ) conditions.Whole-cell radioligand binding assay was performed using [ 3 H]-prazosin. Cell viability was measured by propidium iodide staining and caspase 3/7 assay. Glucose uptake was measured using [ 3 H]-2-deoxy-glucose. Activation of cardioprotective signalling proteins were examined using AlphaScreen SureFire and In-Cell Western assays.Results: Stimulation of α 1A -AR with A61603 increased Ca 2+ mobilisation and glucose uptake in NRVMs, but failed to phosphorylate Akt which is a key protein for insulin-mediated glucose uptake. A61603 (100 nM) significantly decreased the percentage of apoptotic cells under hypoxic conditions (Control 22.0 ± 2.0%, A61603 11.9 ± 1.0%). Hypoxia significantly increased the abundance of α 1 -AR protein (Bmax normoxic 62.3 ± 3.2 fmol/mg, hypoxic 102.5 ± 14.6 fmol/mg protein). The general mTOR complex 1/2 inhibitor KU-0063794 (1 µM) significantly inhibited α 1A -ARmediated glucose uptake by 69% and cell survival by 69% in hypoxic NRVMs, whereas the mTOR complex 1 inhibitor rapamycin (10 nM) had no effect, suggesting that mTOR complex 2 has a key role. Conclusion: These findings indicate that α 1A -ARs are proportionally increased in hypoxia, mediate protective signalling in hypoxic NRVMs, increase Akt-independent glucose uptake and promote cell survival via mTORC2.