Role of CARP as a Bio-Mechanosensor

Abstract

Cardiomyocyte mechanical stretch induces hypertrophic gene expression, however, the mechanisms for this are poorly understood. Cardiac ankyrin repeat protein (CARP) is highly expressed in cardiomyocytes and interacts with the spring domain of sarcomeric titin and is also localized in the nucleus. This dual localization suggests that CARP may couple titin spring mechanics to muscle gene expression. CARP is also expressed in cardiac fibroblasts (CF) and that CARP interacts with the transcription factor GATA4. We hypothesize that CARP is a bio-mechanosensor that upon cell stretch, translocates to the nucleus and interacts with GATA4 to induce gene expression. Methods: Neonatal Rat Ventricular Myocytes (NRVMs) and CF were isolated from 1-2 day old rats and cultured on BioFlex plates. After 2 days, NRVMs were transfected with CARP siRNA (50nM) and/or a GATA4-luciferase vector. NRVMs and CF were stretched 5-10% for 60 min (S60) to 48hr at 1Hz using the Flexcell system. Cells were fixed or lysed for microscopy, western blotting, or luciferase assay. Results: Unstretched NRVMs have predominantly sarcomeric CARP immunostaining with low nuclear CARP. CARP translocates to the nucleus with S60 and remains nuclear up to 48hr stretch. ERK inhibition with U0126 prevented S-60-induced CARP nuclear translocation. NRVM S60 induced GATA4 phosphorylation and increased GATA4-luciferase expression, which are both inhibited with CARP siRNA. NRVM S60 followed by cessation of stretch for 60 min (SC60) resulted in depletion of nuclear CARP. CF show nuclear CARP, which becomes cytoplasmic with S60. CF subjected to S60 followed by SC60 showed re-localization of CARP to the nucleus. Conclusion: We conclude that CARP is involved in stretch-mediated signaling in cardiac myocytes and fibroblasts. Our data further suggest disparate mechano-sensing roles for CARP in these cells types.