Sarcomere alignment is regulated by myocyte shape

Abstract

Cardiac organogenesis and pathogenesis are both characterized by changes in myocyte shape, cytoskeletal architecture, and the extracellular matrix (ECM). However, the mechanisms by which the ECM influences myocyte shape and myofibrillar patterning are unknown. We hypothesized that geometric cues in the ECM align sarcomeres by directing the actin network orientation. To test our hypothesis, we cultured neonatal rat ventricular myocytes on islands of micro-patterned ECM to measure how they remodeled their cytoskeleton in response to extracellular cues. Myocytes spread and assumed the shape of circular and rectangular islands and reorganized their cytoskeletons and myofibrillar arrays with respect to the ECM boundary conditions. Circular myocytes did not assemble predictable actin networks nor organized sarcomere arrays. In contrast, myocytes cultured on rectangular ECM patterns with aspect ratios ranging from 1:1 to 7:1 aligned their sarcomeres in predictable and repeatable patterns based on highly localized focal adhesion complexes. Examination of averaged α-actinin images revealed invariant sarcomeric registration irrespective of myocyte aspect ratio. Since the sarcomere subunits possess a fixed length, this observation indicates that cytoskeleton configuration is length-limited by the extracellular boundary conditions. These results indicate that modification of the extracellular microenvironment induces dynamic reconfiguring of the myocyte shape and intracellular architecture. Furthermore, geometric boundaries such as corners induce localized myofibrillar anisotropy that becomes global as the myocyte aspect ratio increases.

Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness

“…Otherwise, ΣjF c j of 7:1 hPSC-CMs was similar to maximal values reported in other studies of single hPSC-CMs (0.1-500 nN) (45,46), possibly reflecting the use of different force-measuring techniques and improved myofibril alignment. Sarcomere length varies with cell aspect ratio in murine neonatal CMs micropatterned on glass (16,20). However, we observed physiological sarcomere lengths around 2 μm and detected no difference in sarcomere length for aspect ratios from 3:1 to 7:1.…”

“…Matrigel is an extracellular matrix mixture (14), and CMs assume the shape of micropatterns of extracellular proteins printed on surfaces (11,15,16). A rectangular shape of hPSC-CMs with a physiological aspect ratio of 7:1 and area of 2,000 μm 2 engineers a physiological organization of sarcomeres (11), with myofibrils aligned along the main cell axis, as in primary adult murine CMs (17).…”

Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip

“…All animal protocols were approved by the Harvard University Animal Care and Use Committee. Cardiac myocytes were isolated by enzymatically digesting ventricles from 2-d-old Sprague-Dawley rats by using previously published protocols (24,48,57). A custom bioreactor based on a previous design (26) was built to apply cyclic strain to tissues cultured on silicone membranes using a linear motor (LinMot) to stretch as many as 10 samples simultaneously in an incubator (23).…”

Aligned electrospun polymer fibres for skeletal muscle regeneration

“…This can be done by using a scaffold with the relevant topography to induce this behaviour via contact guidance (Bashur et al, 2006;Dalby et al, 2003). It is known that the structural components of the extracellular matrix (ECM) and their interaction with transmembrane proteins, such as integrins play a role in the organisation of tissues, including the organisation of myoblasts (Bray et al, 2008;Schiaffino and Partridge, 2008). These ECM cues provide topology and mechanical properties relevant to the tissue and changes in ECM structure can affect cellular behaviour.…”