Dcm-Causing Mutation E361G in Actin Uncouples Myofibril Ca2+ Sensitivity from Protein Phosphorylation

Abstract

Experiments using the in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation of troponin I and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle myofibrils producing isometric force. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.1 mol Pi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of propranolol which reduced of both TnI bisphosphorylation and MyBP-C phosphorylation by at least 95%. Then we measured contraction in single myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The Ca2+-sensitivity of isometric force and the kinetics of tension development and relaxation for the ACTC E361G TG mouse myofibrils were compared with WT. Modulation of the Ca2+-sensitivity by changes in sarcomere length and by the Ca2+-sensitizer EMD 57033 was further investigated.The maximum isometric force and the tension development rate was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse myofibrils. The ACTC E361G myofibrils had higher Ca2+-sensitivity of force development then WT. Despite that, the magnitude of length dependent activation was well preserved in ACTC E361G myofibrils. Essentially, while the Ca2+-sensitivity of isometric force was increased 1.4-fold when WT myofibrils were dephosphorylated, the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. Additionally, comparing dephosphorylated WT vs WT, the relaxation rate was 1.3-fold slower but dephosphorylation did not change the kinetics of E361G myofibril. Changes in Ca2+-sensitivity were correlated with changes in relaxation rate. Thus the ACTC E361G mutation uncoupled Ca2+-sensitivity and lusitropy from the level of TnI and MyBP-C phosphorylation in intact myofibrils.