Effects Of Silencing Synapse Associated Protein-97 On Cardiac Potassium Currents

Abstract

Introduction: Synapse associated protein-97 (SAP97) is a scaffolding protein expressed in cardiac myocytes. Previous studies have suggested that SAP97 interacts with and modifies properties of ion channels. We have investigated the functional effect of silencing SAP97 on major repolarizing currents in adult rat ventricular myocytes (ARVMs). Methods: SAP97 was silenced using a shRNA expressing adenovirus. The standard patch clamp technique was used to investigate the effects of this silencing on potassium currents in ARVMs. Control experiments were carried out in ARVMs infected with a GFP expressing adenovirus. Results: Western blot analysis showed that SAP97 was silenced in ARVM after 3 days in culture. In SAP97 silenced ARVMs, IK1 density was reduced by ∼50% when measured at -100 mV (Holding potential (HP) = -50 mV). Average current density was -1.85±0.3 pA/pF, n=12 as compared to -3.76±0.5 pA/pF, n = 6 in control cells. Depolarization-activated (at +60 mV, HP = -70 mV) currents in the ARVM were fitted with a two-exponential function for analysis. Amplitude and kinetic analysis of the fits showed that there was a 30% decrease in the current density of the first component in SAP97 silenced ARVMs (9±1.9 pA/pF, n=5) as compared to control (13.5±0.9 pA/pF, n=4). SAP97 silencing however did not significantly change the kinetics of the first component. Time constants averaged 68±12 msec and 1.1±0.1 sec in control versus 121±24 msec and 1.47±0.21 sec in SAP97 silenced cells. Compared to the control cells, there was no change in the current density of the second component in SAP97 silenced cells. Current amplitude averaged 3.1±0.4 pA/pF (n=5) and 3.7±0.2 pA/pF (n=4), respectively for SAP97 silenced ARVMs and control. These results suggest that the silencing of SAP97 has differential effects on potassium currents in adult cardiac myocytes.