Compositions and methods for measuring cellular mechanical stress

#Provided are compositions and methods employing cells encapsulated within and attached to a hydrogel, e.g., for measuring mechanical strain and/or stress of the cell and for investigating the mechano-chemo-transduction mechanisms at cellular and molecular levels.

#It is a fundamental requirement for biological cells to sense and respond to mechanical stress. In the heart, high peripheral resistance (high blood pressure, pressure-overload) is countered by increasing contractility of the myocardium, known as the Anrep effect; enlarged diastolic volume (volume overload) is also countered by increasing contractility, known as the Frank-Starling mechanism. While the heart senses mechanical stress and adjusts contractility to meet varying hemodynamic needs, excessive strain and stress (pressure-overload, volume-overload) also lead to cardiac dysfunction and heart disease development. The mechanical integrity of a cardiac myocyte is maintained by a network of structural proteins including the costamere (elaborated version of focal adhesion complex) that encircles the z-disks to provide structural support (FIG. 1). The costamere comprises the dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin complex (VTI). The importance of these mechanical stress bearing and sensing proteins are demonstrated in various Muscular Dystrophy, in which mutations in DGC or VTI lead to cardiac dysfunction and dilated cardiomyopathy. Although these phenomena are well known for over a century, the cellular and molecular mechanisms that transduce mechanical strain and stress to intracellular biochemical reactions, so called mechano-chemo-transduction (MCT) mechanism, remains poorly understood to date. As result, effective therapies are still lacking for treating mechanical stress-induced heart diseases such as Muscular Dystrophy Cardiomyopathy and Hypertension induced Cardiac Arrhythmias and Heart Failure.

#In one aspect, provided are compositions comprising a cell encapsulated in a hydrogel. In varying embodiments, the hydrogel is comprised of a first binding partner of a binding partner pair or a first binding partner of the binding partner pair conjugated to a biocompatible polymer, wherein the first binding partner is cross-linked with Y—X—Y crosslinker, wherein X is a linear hydrophilic polymer and Y is a second binding partner of the binding partner pair, and wherein the cell is attached directly or indirectly to the first binding partner. In some embodiments, the hydrogel is comprised of a first binding partner of a binding partner pair conjugated to a biocompatible polymer, wherein the biocompatible polymer is cross-linked with a boronate-X-boronate crosslinker, wherein X is a linear hydrophilic polymer, and wherein the cell is attached directly or indirectly to the first binding partner. In varying embodiments, the binding partner pair is selected from the group consisting of avidin/biotin, glutathione/glutathione S-transferases (GST), maltose-binding protein (MBP)/maltose, Histag/Imidazole plus Zn2+, and vancomycin/D-alanine-D-alanine (DADA), wherein either member of the binding partner pair can be the first binding partner or the second binding partner. See, e.g., Rao, et al., Science. (1998) 280(5364):708-11. Additional binding partner pairs of use are described, e.g., in Lichty, et al., Protein Expression and Purification (2005) 41:98-105.

#FIG. 1 illustrates a muscle cell structure and the costamere complex (green beams in the upper panel) that encircles the z-line to provide structural support. Costamere comprises dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin complex (VTI). Our studies show mechano-chemo-transduction (MCT) pathways from DGC and VTI to nitric oxide synthase 1 (or nNOS) and nitric oxide synthase 3 (or eNOS) and to Ca2+ handling proteins.

#FIG. 2 illustrates the Cell-in-Gel system used in an experiment. The solution is continuously perfused to maintain the physiological condition (pH, temperature, ionic milieu etc.) in the hydrogel. Electric field is used to stimulation myocyte contraction. Microscopy is used to image the myocyte contraction and the mechano-chemo-transduction signaling events at cellular and molecular levels.

#FIG. 3 illustrates DGC and VTI complex in a costamere (modified from Guo et al., (2011) Journal of Molecular and Cellular Cardiology. 50:606-12).

#1. Introduction

#The compositions, devices, systems, methods and kits described herein enhance the understanding of the mechano-chemo-transduction (MCT) mechanism. They achieve at least two important capabilities: one is to control mechanical stress at the single cell level in 3-D environment mimicking the myocardium; the other is to tug on specific cell-surface mechanosensors during myocyte contraction. These technical capabilities are useful for interrogating the MCT mechanisms at the cellular and molecular levels. In contrast to the present compositions, devices, systems, methods and kits, currently available techniques do not have both capabilities.

#Provided herein is a ‘Cell-in-Gel’ system to embed single live cells, including myocytes, in a 3D elastic gel that mimics the in vivo mechanical environment. This system differs from the currently used techniques in two important ways. First, the cell (e.g., myocyte) contracts against the elastic gel which imposes not only longitudinal tension and transverse compression on the cell but also shear and normal stresses on the cell surface. Second, the Cell-in-Gel system enables tethering of specific mechanosensors on the cell surface (e.g., integrins, glycoproteins) to the gel matrix to interrogate how the MCT complexes transduce mechanical stress to affect intracellular Ca2+ dynamics.