Human organ systems require temporally and spatially coordinated multicellular actions at a macroscale to actuate, sustain, or terminate dedicated and vital functions. Cells that comprise discrete or distributed physiologic systems that fail to respond to appropriate stimuli with coordination may cause significant morbidity and often mortality. Collective and coordinated physiologic activities typically involve millions to billions of cells that may span large physical distances. Technologies for quantifying the electrical, chemical, and mechanical coupling in these multicellular systems are critically important to understanding the underlying mechanisms of disease and develop therapeutic approaches. However, no technology currently exists to quantify rapid mechanical cell responses to transmitted distal perturbations for all cells within a collection of cells. This multi- PI proposal (Chiou (contact PI) and Teitell) aims to develop a new platform imaging technology called SPOT (single pixel optical technology) for concurrent and direct measurements of cellular traction forces over a 1.0 x 1.0 cm2 field of view (FOV) with cellular spatial resolution, and a 1,000 frames/sec temporal resolution. SPOT provides a 4-order of magnitude larger FOV than conventional traction force microscopy. Cardiomyocytes (CMs) are the test bed here because of a high potential for impact in cardiovascular disease, the leading cause of mortality in the Western World. We will demonstrate the ability for SPOT to determine quantitative indices of abnormalities for human CM contraction and relaxation in healthy and diseased states. We will establish proof of concept studies in SPOT screens for small molecules that augment or affect CM contraction in desmoplakin deficient states. We will build a platform that integrates SPOT for direct contraction force measurements and Optical Mapping for electrical property measurements for sheets of CMs. This will enable, for the first time, studies of temporal and spatial electromechanical coupling behaviors for sheets of CMs at single cell resolution. We will distinguish different subtypes of CMs, their distributions, their interactions, and their phenotypic responses under external perturbations. And we will apply this platform to investigate the structural and electromechanical coupling properties of hESC-derived CMs by integrating quantitative biomass and stiffness data measured using non-invasive live cell interferometry (LCI). Changes in biomass and cell stiffness are druggable biophysical parameters with correlates to mechanical contraction/relaxation cycles of CMs. In addition to detailed studies of CMs that have the potential to impact the number one killer of US citizens, SPOT applications should have utility and provide new insights in additional settings that require cell or tissue traction-force generation. Such settings could include models in a dish for wound healing, cancer cell metastasis, or models of diseases that affect cell and tissue structural integrity, such as connective tissue disorders Ehlers-Danlos or Marfan syndromes.
PROJECT NARATIVE Our proposal is exclusively technology development but portends public health relevance because we will invent a way to quantify previously undiscoverable interactions and mechanical responses to external and internal perturbations in interconnected biological systems, as occurs in physiologic and pathologic states. We will develop, test and fine-tune a new technology platform called SPOT (Single Pixel Optical Technology) to extract mechanical responses at cellular resolution in a very wide field, in real-time, concurrently for all cells in a sheet to enable studies and potentially new-age therapeutics that are currently impossible.
