Multiscale Characterization of Engineered Cardiac Tissue Architecture

Abstract

In a properly contracting cardiac muscle, many different subcellular structures are organized into an intricate architecture. While it has been observed that this organization is altered in pathological conditions, the relationship between length-scales and architecture has not been properly explored. In this work, we utilize a variety of architecture metrics to quantify organization and consistency of single structures over multiple scales, from subcellular to tissue scale as well as correlation of organization of multiple structures. Specifically, as the best way to characterize cardiac tissues, we chose the orientational and co-orientational order parameters (COOPs). Similarly, neonatal rat ventricular myocytes were selected for their consistent architectural behavior. The engineered cells and tissues were stained for four architectural structures: actin, tubulin, sarcomeric z-lines, and nuclei. We applied the orientational metrics to cardiac cells of various shapes, isotropic cardiac tissues, and anisotropic globally aligned tissues. With these novel tools, we discovered: (1) the relationship between cellular shape and consistency of self-assembly; (2) the length-scales at which unguided tissues self-organize; and (3) the correlation or lack thereof between organization of actin fibrils, sarcomeric z-lines, tubulin fibrils, and nuclei. All of these together elucidate some of the current mysteries in the relationship between force production and architecture, while raising more questions about the effect of guidance cues on self-assembly function. These types of metrics are the future of quantitative tissue engineering in cardiovascular biomechanics.

Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs

“…Substrates were fabricated for structural studies as described previously [5,8,19,24,46]. Briefly, large cover glass (Brain Research Laboratories, Newton, MA) was cleaned by sonicating, then spin coated with 10:1 Polydimethylsiloxane (PDMS; Ellsworth Adhesives, Germantown, WI).…”

“…2013-3093). Cardiomyocytes were then isolated from the ventricular myocardium as described previously [5,8,19,24]. Briefly, after rinsing the ventricular tissue in Hanks' balanced salt solution buffer (HBSS; Life Technologies, Carlsbad, CA), the tissues were incubated overnight (12 h) at 4˚C in a 1 mg/mL trypsin solution (Sigma Aldrich, Inc., Saint Louis, MO) dissolved in HBSS.…”

“…Assessment of cellular morphology and structure is fundamental to the study of striated muscles. It has been used to characterize the developmental stage [1][2][3], engineered tissues [4][5][6][7][8], effects of disease [9][10][11][12] or injury [13][14][15][16], and treatment with pharmacological agents [17] as well as used to predict function [18][19][20]. Indeed, the ability of striated muscle cells to contract is dependent on the nearly crystalline order of its cytoskeletal components [21,22], which makes evaluation of structure paramount.…”

“…In particular, because the uniaxial force of sarcomeres is maximized when they are all oriented in the same direction, the orientational order of the z-line protein α-actinin has been used extensively as a metric [3,6,18,30,36]. Similarly, the correlation between the orientational order of z-lines and actin fibrils is also an important metric because the relative orientation of these structures transitions from parallel to perpendicular during development [5,8,37,38]. The relative location and lateral alignment of z-lines in neighboring myofibrils is also hypothesized to influence contractile function and to be an important metric to assess myofibril formation [20,[32][33][34][39][40][41][42].…”

Special Issue: Spotlight on the Future of Cardiovascular Engineering:Frontiers and Challenges in Cardiovascular Biomechanics

“…The microvessel platform allows precise control of factors such as ECM material, cell types, flow rates, pressures, and biochemical environment to study endothelial-blood interactions, endothelial-perivascular interactions, angiogenesis, and tumor biology. Also in this special issue, Drew et al used engineered cardiac tissue to investigate self-assembly and force development in cardiomyocytes [3]. They found that self-assembly and force generation are optimized under similar conditions and that the organizational structure of various cytoskeletal elements is tightly correlated.…”