Phosphatidylinositol 4,5-bisphosphate regulates CapZβ1 and actin dynamics in response to mechanical strain

Abstract

Mechanical stress causes filament remodeling leading to myocyte hypertrophy and heart failure. The actin capping protein Z (CapZ) tightly binds to the barbed end of actin filaments, thus regulating actin assembly. The hypothesis is that the binding between CapZ and the actin filament is modulated through phosphatidylinositol 4,5-bisphosphate (PIP2) and how the COOH-terminus of CapZβ1 regulates this binding. Primary neonatal rat ventricular myocytes (NRVMs) were strained at 10% amplitude and 1-Hz frequency. Dot blotting measured the PIP2 amount, and affinity precipitation assay assessed the direct interaction between PIP2 and CapZβ1. Fluorescence recovery after photobleaching of green fluorescent protein-CapZβ1 and actin-green fluorescent protein after 1 h of strain shows the dynamics significantly increased above the unstrained group. The increases in CapZ and actin dynamics were blunted by neomycin, suggesting PIP2 signaling is involved. The amount of PIP2 dramatically increased in NRVMs strained for 1 h. With a ROCK or RhoA inhibitor, changes were markedly reduced. Subcellular fractionation and antibody localization showed PIP2 distributed to the sarcomeres. More PIP2-bound CapZβ1 was found in strained NRVMs. Less PIP2 bound to the CapZβ1 with its COOH-terminus intact than in the COOH-terminal mutant of CapZβ1, suggesting some inhibitory role for the COOH-terminus. Myocyte hypertrophy normally induced by 48 h of cyclic strain was blunted by dominant negative RhoA or neomycin. This suggests that after many hours of cyclic strain, a possible mechanism for cell hypertrophy is the accumulation of thin filament assembly triggered partially by the increased PIP2 level and its binding to CapZ.