Human mutations that truncate the massive sarcomere protein titin (TTNtv) are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtv, diminish contractile performance and are pathogenic. By combining functional analyses with RNAseq, we explain why truncations in the A-band domain of TTN cause DCM while truncations in the I-band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS-cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodelling.

Interstitial flow is the convective transport of fluid through tissue extracellular matrix. This creeping fluid flow has been shown to affect the morphology and migration of cells such as fibroblasts, cancer cells, endothelial cells, and mesenchymal stem cells. A microfluidic cell culture system was designed to apply stable pressure gradients and fluid flow and allow direct visualization of transient responses of cells seeded in a 3D collagen type I scaffold. We used this system to examine the effects of interstitial flow on cancer cell morphology and migration and to extend previous studies showing that interstitial flow increases the metastatic potential of MDA-MB-435S melanoma cells [Shields J, et al. (2007) Cancer Cell 11:526-538]. Using a breast carcinoma line (MDA-MB-231) we also observed cell migration along streamlines in the presence of flow; however, we further demonstrated that the strength of the flow as well as the cell density determined directional bias of migration along the streamline. In particular, we found that cells either at high seeding density or with the CCR-7 receptor inhibited migration against, rather than with the flow. We provide further evidence that CCR7-dependent autologous chemotaxis is the mechanism that leads to migration with the flow, but also demonstrate a competing CCR7-independent mechanism that causes migration against the flow. Data from experiments investigating the effects of cell concentration, interstitial flow rate, receptor activity, and focal adhesion kinase phosphorylation support our hypothesis that the competing stimulus is integrin mediated. This mechanism may play an important role in development of metastatic disease. mechanobiology | computational model | signaling | cell mechanics T issues are composed of cells residing in an extracellular matrix (ECM) containing interstitial fluid that transports nutrients and signaling molecules (1, 2). Osmotic and hydrostatic pressure gradients across tissues resulting from physiologic processes such as drainage toward lymphatics, inflammation, locally elevated pressures due to tumor growth or leaky microvessels, and muscle contraction each drive fluid flow through the ECM (2, 3). This fluid flow is termed interstitial flow and has long been recognized to be instrumental in tissue transport and physiology (1, 4, 5). Chary and Jain used fluorescence recovery after photobleaching to directly observe fluid flow in the tissue interstitium and determined typical flow velocities to be on the order of 0.1-2.0 μm/s, and more recent studies have demonstrated that flow can reach velocities as high as 4.0 μm/s (6, 7).Interstitial flow is particularly important in driving transport in the vicinity of tumors, as neoplastic tissue is often characterized by localized increases in interstitial pressure, leading to high interstitial pressure gradients at the tumor margin (8). Interstitial flow has hence emerged as a possible stimulus for guiding tumor cell migration in the formation of metastases (9-12).Shields et al. observed increase...

Current measurements of the biomechanical properties of cells require physical contact with cells or lack sub-cellular resolution. Here, we developed a label-free optical microscopy technique based on Brillouin light scattering capable of measuring intracellular longitudinal modulus with optical resolution. We obtained 3D Brillouin maps of cells in 2D and 3D microenvironments, which reveal mechanical changes due to cytoskeletal modulation and cell volume regulation.

Forces generated by cells are critical regulators of cell adhesion, signaling and function, and are essential drivers in the morphogenetic events of development. Over the past 20 years, several methods have been developed to measure these forces. Despite recent substantial interest in understanding the contribution of these forces in biology, implementation and adoption in the broader biological community remains challenging due to the inherently multidisciplinary expertise required to conduct and interpret these measurements. In this review, we introduce the established methods, and highlight the technical challenges associated with implementing each technique in a biological laboratory.