Inhibition of Akt Attenuates Isoproterenol-Induced RyR-Dependent Diastolic Ca2+ Release in Rabbit Ventricular Myocytes

Abstract

Cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca2+ release is increased by beta-adrenergic receptor (ß-AR) stimulation. Increased diastolic RyR activity can lead to arrhythmogenic spontaneous SR Ca2+ release. Our studies have shown that calmodulin-dependent protein kinase II (CaMKII) activation by nitric oxide (NO) implicates nitric oxide synthase (NOS) in the pathway. Here we investigate the role Akt may play in activating NOS, thus increasing diastolic SR Ca2+ release during ß-AR stimulation in isolated rabbit ventricular myocytes. Western blot analysis showed a dose-dependent increase in phosphorylated Akt in response to the β-AR agonist isoproterenol (ISO). Cytosolic Ca2+ was measured using fluo-4 loaded intact myocytes. SR Ca2+ was varied by field-stimulated to steady state at different frequencies. Tetracaine (1mM) was used to rapidly and reversibly block the RyR. The tetracaine-dependent shift of Ca2+ from the cytosol to the SR is proportional to diastolic Ca2+ release. Primary myocytes were treated with 250nM ISO with and without Akt inhibitor-X (AIX; 5μM; 30min pre-incubation and continuous perfusion). ISO-induced increase in the diastolic SR Ca2+ release was abolished by the treatment of myocytes with AIX. When data were selected such that SR[Ca2+] was matched in each group (ISO: 155.22±5.1μM; ISO+AIX: 153.92±3.8μM), myocytes treated with ISO had significantly higher tetracaine-dependent increase in diastolic SR[Ca2+] (12.43±3.8μM) vs. those treated with ISO and AIX (1±2.2μM) (P=0.01, t-test). The results suggest that the ISO-dependent increase in diastolic SR Ca2+ release is dependent upon Akt. To further test this hypothesis myocytes were cultured for 24 hours with adenovirus to express a dominant-negative Akt construct (Akt-dn). Preliminary evidence shows reduced diastolic SR[Ca2+] release for a given SR[Ca2+] in Akt-dn myocytes. This evidence indicates that Akt activation may be an important upstream effector involved in the β-AR-induced diastolic SR Ca2+ release.