Myofibrillar architecture in engineered cardiac myocytes

Abstract

Morphogenesis is often considered a function of transcriptional synchrony and the spatial limits of diffusing mitogens; however, physical constrainment by the cell microenvironment represents an additional mechanism for regulating self-assembly of subcellular structures. We asked whether myocyte shape is a distinct signal that potentiates the organization of myofibrillar arrays in cardiac muscle myocytes. We engineered the shape of neonatal rat ventricular myocytes by culturing them on microfabricated fibronectin islands, where they spread and assumed the shape of the island. Myofibrillogenesis followed, both spatially and temporally, the assembly of unique actin networks whose architecture was predictable given the shape of the island. Subsequently, the z lines of the sarcomeres aligned and registered in distinct patterns in different regions of the myocytes in such a way that orthogonal axes of contraction could be distinctly engineered. These data suggest that physical constrainment of muscle cells by extracellular matrix may be an important regulator of myofibrillar organization.

Fig. 1

Neonatal cardiac myocytes immunostained against sarcomeric α-actinin. A) in vivo morphology; B) pleomorphic on an unpatterned, FN-coated surface; C) on a micropatterned FN rectangle; D) star; E) triangle. All images are single, mononucleated ventricular myocytes. Patterned myocytes were cultured for 48 hours. Scale bar is 10 microns.

Fig. 2

Neonatal cardiac myocyte cultured on a micropatterned FN island for 48 hrs. Left) Flourescent micrograph of immunostained sarcomeric α-actinin; Right) Flourescent micrograph of actin stained with FITC-conjugated phalloidin. Scale bar is 10 microns.

Fig. 3

Cardiac myocyte cultured on a 50 μm square FN island for 72 hrs. Left) Phase image, with membrane wrinkles apparent in the corners; Right) Flourescent image revealing stained sarcomeric α-actinin at the Z-lines. Scale bar is 10 microns.