Abstract 10720: Inhibition of Akt Reverses the Isoproterenol-enhanced Diastolic Calcium Leak in Rabbit Ventricular Myocytes
Cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca2+ leak is increased in heart failure and during beta-adrenergic (β-AR) stimulation. This increased SR Ca2+ leak could limit the SR [Ca2+] thereby decreasing contractility in HF and could also lead to arrhythmogenic Ca-dependent inward depolarizing current resulting in HF. It has recently been shown in our lab that this leak is dependent on calcium-calmodulin-dependent protein kinase II (CaMKII) and subsequent NOS activation, and that it is independent of either protein kinase A (PKA) activation or an increase in free Ca2+ concentration in the cytosol ([Ca2+]i). Here we investigate whether Akt is also involved in the pathway leading to increased diastolic leak during β-AR stimulation.
Methods: [Ca2+]i was measured using fluo-4 in left ventricular myocytes isolated from rabbits. To vary the SR load, myocytes were 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 (decrease in [Ca2+]i and increase in SR Ca2+ content) is proportional to SR Ca2+ leak. To activate β-AR, isoproterenol (ISO; 250 nM) was added to perfuse the myocytes starting 5 min before leak assessment through the end.
Results: ISO-induced increase in the SR Ca2+ leak is abolished by the treatment of myocytes with Akt inhibitor X (AIX; 5μM; 30min pre-incubation and perfusion throughout the experiment). When SR Ca2+ load was matched in each group (ISO alone: 155.22 ± 5.1 μM; ISO + AIX: 153.92 ± 3.8 μM), myocytes treated with ISO alone had significantly higher leak (12.43 ± 3.8 μM) vs. those treated with ISO and AIX (1 ± 2.2 μM) (P = 0.01, t-test). This evidence indicates that Akt activation may also be involved in the β-AR-induced SR Ca2+ leak possibly upstream from CaMKII and NOS activation.
- Ventricular function
- Beta-adrenergic receptor agonists
- Diastolic function
- Receptor-mediated signaling
- © 2011 by American Heart Association, Inc.