Abstract 2328: Overexpression of Ito Channel Kv4.3 Resets ICa, L and Shifts Its Regulation From CaMKII to PKA
Background: We have recently shown that Ito channel Kv4.3 overexpression reduces cardiac CaMKII activity and that acute CaMKII inhibition reduces L-type Ca2+ current (ICa) in myocytes. Here, we tested the effect of in vivo Kv4.3 expression on ICa in ventricular myocytes.
Methods: Male C57BL6 mice (6 weeks of age) were injected with Ad-Kv4.3 or Ad-beta-gel (as control) in the left ventricle (LV). These adenoviruses contain GFP fusion protein constructs. 7 days after adenovirus injection, left ventricular myocytes were isolated from these mice and used for ICa recording using whole-cell patch clamp at room temperature (the green myocytes under florescence microscope were selected for ICa recording) and Western blot analysis.
Results: The Kv4.3 transfection rate in mouse LV is 30 – 40%. Western blot showed an unchanged CaMKII level but a significantly reduced CaMKII autophosphorylation (using anti-Phospho-CaMKII antibody) and phospholamban (PLB)-Thr17 phosphorylation (using anti-PLB-Thr17 antibody) in the Ad-Kv4.3 transfected myocytes, indicating a reduced CaMKII activity. Interestingly, ICa density was significantly increased in the Ad-Kv4.3 transfected myocytes compared with control myocytes without changing the voltage dependence of ICa activation. The peak ICa (recorded at +10 mV, holding potential −50 mV) was 8.6±0.45 pA/pF in Ad-Kv4.3 transfected myocytes (n=55) and 6.8±0.2 pA/pF in control myocytes (n=38), respectively (p<0.05). In addition, the ICa inactivation (current decay) time course was significantly slowed in Ad-Kv4.3 transfected myocytes (Tau fast was 8.2±0.4 ms and Tau slow was 81.6±0.3 ms) compared with control (Tau fast was 7.2±0.4 ms and Tau slow was 71.2±0.5 ms, p<0.05). The slowed inactivation indicates an increased channel phosphorylation by PKA. Indeed, Western blot analysis showed a significantly increased PKA expression level and an increased PLB-Ser16 phosphorylation. In addition, the expression of Ca2+ channel alpha-subunits Cav1.2 was significantly increased in Ad-Kv4.3 transfected myocytes compared with control.
Conclusion: Kv4.3 overexpression in ventricular myocytes causes a significant CaMKII inhibition which resets ICa and shifts its regulation from CaMKII to PKA.