Cell Signaling in Cardiac Myocytes
In cardiac muscle, the interplay between forces generated by the contractile apparatus and sensed by integrins is thought to play an important role in cardiac remodeling and the pathogenesis of diseases such as hypertrophic cardiomyopathy (HCM). Although the effects of external forces on integrin signaling have been extensively studied in cardiac myocytes, the effects of internal contractile forces on integrin signaling remain undefined. To study the effects of altered contractility on integrin signaling, we have developed an in vitro tissue model consisting of a flexible micropatterned scaffold which facilitates organized growth and differentiation of neonatal rat ventricular myocytes (NRVMs) into an adult phenotype with an organized filament structure (cardiofilaments) with intercalated discs.
To measure integrin activation in this model system, we have constructed an adenovirus expressing a talin-GFP fusion protein. Binding of this talin-GFP to integrins upon integrin activation will be detected using total internal reflectance (TIRF) microscopy. We have confirmed talin-GFP colocalization with integrins at cell-ECM contacts using confocal microscopy. To measure traction forces generated by cardiofilaments, we use TIRF microscopy to measure displacements of fluorescent markers embedded within the laminin matrix. Simultaneous measurements of integrin activation and cardiofilament mechanics will allow us to directly determine how contractile forces altered by small molecule effectors such as blebbistatin influence integrin signaling. We hypothesize that activating sarcomere contractile forces will increase traction forces and activate integrins, initiating a hypertrophic response.
- Milad Motarjemi, 2011