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Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography.

ABSTRACT: Cellular structure and function are interdependent. To understand this relationship in beating heart cells, individual neonatal rat ventricular myocytes (NRVMs) were analyzed one and 3 days after plating when cultured on different stiffness (100, 400 kPa) and surface structures (flat or [Formula: see text] high, [Formula: see text] diameter, microposts spaced [Formula: see text] apart) manufactured from polydimethylsiloxane. Myofibril structure seen by immunohistochemistry was organized in three dimensions when NRVMs were attached to microposts. On day three, paxillin distribution near the post serving as cellular anchorage was quantified on both soft posts (12.04 % of total voxel count) and stiff posts (8.16 %). Living NRVMs were analyzed using line scans for sarcomeric shortening and shortening velocity, and traction force microscopy for surface stress and surface tension. One day after plating, NRVMs shortened more on soft posts ([Formula: see text] at [Formula: see text]) compared to either soft flat ([Formula: see text] at [Formula: see text]), stiff posts ([Formula: see text] at [Formula: see text]) or stiff flat ([Formula: see text] at [Formula: see text]). NRVMs have decreased shortening and shortening velocity on soft posts ([Formula: see text] at [Formula: see text]) compared to soft flat ([Formula: see text] at [Formula: see text]) substrates. The surface stress and surface tension increased over time for both soft post ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) and flat ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) substrates. Paxillin displacement during contraction on day three was significantly greater in NRVMs attached to soft posts [Formula: see text] compared to flat [Formula: see text] substrates. The volume and time creating four-dimensional data, interpreted by structural engineering theory, demonstrate subdomain structure is maintained by the counterbalance between the external load acting upon and the internal forces generated by the cardiomyocyte. These findings provide further insight into localized regulation of cellular mechanical function.

SUBMITTER: Broughton KM

PROVIDER: S-EPMC4383742 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography.

Broughton Kathleen M KM Russell Brenda B

Biomechanics and modeling in mechanobiology 20141002 3

Cellular structure and function are interdependent. To understand this relationship in beating heart cells, individual neonatal rat ventricular myocytes (NRVMs) were analyzed one and 3 days after plating when cultured on different stiffness (100, 400 kPa) and surface structures (flat or [Formula: see text] high, [Formula: see text] diameter, microposts spaced [Formula: see text] apart) manufactured from polydimethylsiloxane. Myofibril structure seen by immunohistochemistry was organized in three ...[more]

PMID: 25273278

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Project description:The measurement of the contractile behavior of single cardiomyocytes has made a significant contribution to our understanding of the physiology and pathophysiology of the myocardium. However, the isolation of cardiomyocytes introduces various technical and statistical issues. Traditional video and fluorescence microscopy techniques based around conventional microscopy systems result in low-throughput experimental studies, in which single cells are studied over the course of a pharmacological or physiological intervention. We describe a new approach to these experiments made possible with a new piece of instrumentation, the CytoCypher High-Throughput System (CC-HTS). We can assess the shortening of sarcomeres, cell length, Ca2+ handling, and cellular morphology of almost 4 cells per minute. This increase in productivity means that batch-to-batch variation can be identified as a major source of variability. The speed of acquisition means that sufficient numbers of cells in each preparation can be assessed for multiple conditions reducing these batch effects. We demonstrate the different temporal scales over which the CC-HTS can acquire data. We use statistical analysis methods that compensate for the hierarchical effects of clustering within heart preparations and demonstrate a significant false-positive rate, which is potentially present in conventional studies. We demonstrate a more stringent way to perform these tests. The baseline morphological and functional characteristics of rat, mouse, guinea pig, and human cells are explored. Finally, we show data from concentration response experiments revealing the usefulness of the CC-HTS in such studies. We specifically focus on the effects of agents that directly or indirectly affect the activity of the motor proteins involved in the production of cardiomyocyte contraction. A variety of myocardial preparations with differing levels of complexity are in use (e.g., isolated muscle bundles, thin slices, perfused dual innervated isolated heart, and perfused ventricular wedge). All suffer from low throughput but can be regarded as providing independent data points in contrast to the clustering problems associated with isolated cell studies. The greater productivity and sampling power provided by CC-HTS may help to reestablish the utility of isolated cell studies, while preserving the unique insights provided by studying the contribution of the fundamental, cellular unit of myocardial contractility.

Dataset Information

Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography.

Publications

Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography.

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