Fig. 2.
Role of SAP97 in the reciprocal modulation of IK1 and INa. (A and B) Ad-shSAP97 infection silences SAP97 expression in adult rat ventricular myocytes (ARVMs) by day 3 in culture compared with control. (A) Immunoblot showing immunodetection of SAP97 expression (Top and Middle) and GAPDH (loading control; Bottom) from cells infected with either a control (Ad-515) or shSAP97 (Ad-shSAP97) adenovirus. Middle (SAP97) shows a longer exposure of the same immunoblot shown in Top; the overexposure confirms expression of SAP97 in the Ad-SAP97–silenced cells and shows the contrasting intensities and apparent protein levels between control and SAP97-silenced ARVMs. (B) Densitometric analysis of SAP97 expression normalized to GAPDH levels in control (black) and shSAP97-silenced (red) cells. Values represent data (mean ± SEM) from two different preparations harvested 3 d after infection. SAP97 expression was effectively knocked down by ∼56% on day 3. (C) IK1 density is reduced after SAP97 silencing in ARVMs. Peak inward current density at −100 mV (control: −7.67 ± 1.52 pA/pF; Ad-shSAP97: −4.55 ± 0.47 pA/pF) and peak outward current density at −60 mV (control: 1.03 ± 0.46 pA/pF; Ad-shSAP97: 0.34 ± 0.03 pA/pF) were significantly different (P = 0.02 and P = 0.04, respectively) between myocytes infected with shSAP97 (N = 4, n = 11) and myocytes infected with Ad-515 (control; N = 2, n = 6). Inset shows the protocol used to measure the current. (D) Effects of SAP97 knockdown on sodium channel current density in ARVMs. Superimposed INa density/voltage relationships in control (Ad-515) and SAP97-silenced (Ad-shSAP97) cells. A significant reduction in INa density was observed for SAP97-silenced cells at several tested voltages. For both control and silenced conditions, N = 2 and n = 11. (Inset) Voltage clamp step protocol. (E) Voltage-dependent activation and inactivation (m∞ and h∞) curves for the data presented in D. *P < 0.005; #P < 0.05; δP < 0.01 (unpaired t test with Welch’s correction).