PKC epsilon signaling effect on actin assembly is diminished in cardiomyocytes when challenged to additional work in a stiff microenvironment.

Abstract

The stiffness of the microenvironment surrounding a cell can result in cytoskeletal remodeling, leading to altered cell function and tissue macrostructure. In this study, we tuned the stiffness of the underlying substratum on which neonatal rat cardiomyocytes were grown in culture to mimic normal (10 kPa), pathological stiffness of fibrotic myocardium (100 kPa), and a nonphysiological extreme (glass). Cardiomyocytes were then challenged by beta adrenergic stimulation through isoproterenol treatment to investigate the response to acute work demand for cells grown on surfaces of varying stiffness. In particular, the PKCɛ signaling pathway and its role in actin assembly dynamics were examined. Significant changes in contractile metrics were seen on cardiomyocytes grown on different surfaces, but all cells responded to isoproterenol treatment, eventually reaching similar time to peak tension. In contrast, the assembly rate of actin was significantly higher on stiff surfaces, so that only cells grown on soft surfaces were able to respond to acute isoproterenol treatment. Förster Resonance Energy Transfer of immunofluorescence on the cytoskeletal fraction of cardiomyocytes confirmed that the molecular interaction of PKCɛ with the actin capping protein, CapZ, was very low on soft substrata but significantly increased with isoproterenol treatment, or on stiff substrata. Therefore, the stiffness of the culture surface chosen for in vitro experiments might mask the normal signaling and affect the ability to translate basic science more effectively into human therapy.