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 alpha-actinin images revealed invariant sarcomeric registration irrespective of myocyte aspect ratio. Since the sarcomere sub-units 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.

Figure 1

Distribution of actin (A,C) α-actinin (B) and vinculin (D) in pleomorphic cultured myocytes. One example myocyte is shown in (A) and (B), a different myocyte is shown in (C) and (D). Note the lack of myofibrillar and sarcomeric organization and multiple myofibril axes for both myocytes shown. Scale bar: 10 μm.

Figure 2

(A) A DIC image of a cultured myocyte on a microcontact printed circular ECM island (radius: 26 μm) with the nucleus colored in blue. (B) FAs linking the myocyte to the ECM are highlighted by stained vinculin. The associated F-actin (C) and sarcomeric α-actinin (D) in a representative circular myocyte shows the absence of a preferential axis of organization. The resultant sarcomere organization for an ensemble of circular myocytes is illustrated via averaged images of F-actin (E) and sarcomeric α-actinin (F) distributions from fixed and stained myocytes. Scale bars: 10 μm.

Figure 3

Cardiac myocytes on rectangular microcontact printed (μCP) ECM islands, FAs highlighting the myocyte-ECM contacts, and cytoskeletal architecture and averaged myofibrillar organization. Three cellular aspect ratios are shown: (A) 1:1, (B) 2:1, and (C) 3:1. A DIC image and immunofluorescent stains for vinculin, F-actin and sarcomeric α-actinin of a representative cardiac myocyte on a μCP ECM island are shown in panels (i)-(iv), respectively. The averaged distribution of F-actin for each cellular aspect ratio is shown in panel (v).

Figure 4

Cardiac myocytes on rectangular microcontact printed (μCP) ECM islands, FAs highlighting the myocyte-ECM contacts, and cytoskeletal architecture and averaged myofibrillar organization. Two cellular aspect ratios are shown: (A) 5:1 and (B) 7:1. A DIC image and immunofluorescent stains for vinculin, F-actin and sarcomeric α-actinin of a representative cardiac myocyte on a μCP ECM island are shown in panels (i)–(iv), respectively. The averaged distribution of F-actin for each cellular aspect ratio is shown in panel (v). (C) Anisotropy of the myofibrillar network as a function of the myocyte shape. Bars are given as mean ± SEM of the angular standard error of the actin cytoskeleton orientation. Statistically significant differences were found between the circular and 1:1 myocytes as compared to the remaining shapes, as well as between 2:1 myocytes and myocytes with AR > 5:1.

Figure 5

(A) Average distribution of sarcomeric α-actinin from fixed and stained rectangular myocytes. Each myocyte was registered to a uniform coordinate system, normalized and the pixel intensity was averaged over all myocytes. Scale bar is 10 μm for all panels. (B) Intensity profiles from the averaged images as a function of distance along the black lines illustrated in (A). The lines are offset in the y-axis for clarity.