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Modeling the Cardiac Extracellular Matrix to Identify Age-specific Modulators of Cardiomyocyte Function

  • Author(s): Kauss, Mara Ariel
  • Advisor(s): Black, Brian
  • et al.
Abstract

The extracellular matrix (ECM) is a complex network of proteins that surrounds cells in all tissues, providing structural and biochemical cues that influence a multitude of cellular functions. In the heart, the ECM provides the local support and context for highly dynamic cardiomyocytes that are continuously contracting to pump blood throughout the body. However, the cardiac phenotype changes drastically throughout fetal development and postnatal maturation. Fetal cardiomyocytes are small, proliferative, glycolytic cells with disorganized sarcomeric structures. In the adult, however, cardiomyocytes become elongated, aligned, post-mitotic cells that rely on oxidative respiration and have highly organized sarcomeres. These ontogenic changes provide a dramatic increase in contractile efficiency, but sacrifice the heart’s proliferation-dependent ability to regenerate after injury. As a result, heart disease is a major cause of death worldwide, and has warranted a wealth of research efforts to model cardiac tissue function and disease. During cardiac development and maturation, the ECM also undergoes age-dependent changes that can impact various cardiac functions, but the extent of its influence is poorly understood. To model the age-specific cardiac ECM in vitro, we created cell-derived matrices (CDMs) from cardiac fibroblasts to provide the structural, biophysical and chemical context of cardiac-specific ECM. In doing so, we sought to avoid certain caveats of established CDM protocols, which enhance matrix adhesion and thickness via introduction and promotion of singular matrix proteins, skewing the matrix composition, and confounding comparisons made between CDMs. We, therefore, developed a protocol that enhances matrix adhesion and deposition, respectively, by combining an L-polydopamine coating and macromolecular crowding to produce CDMs composed of cell-produced ECM. This methodology was applied to the study of age-dependent phenotypic and functional changes observed in the heart by comparing the morphologic, metabolic and proliferative responses of cardiomyocytes to CDMs produced by fetal and adult cardiac fibroblasts. Additionally, mass spectrometry proteomics identified the enrichment of ECM proteins in fetal and adult CDMs, enabling loss-of-function studies via genetic silencing of enriched proteins during CDM production. Using this strategy, we identified collagen VI as necessary for maximal oxidative respiration in stem cell-derived cardiomyocytes, which is a key component of cardiomyocyte maturation. Furthermore, we conducted a screen of all age-specific enriched CDM proteins for their influence over DNA synthesis in cardiomyocytes, and found that laminin α2, plasmin-mediated signaling in fibroblasts, and fibrosis-associated proteins exerted inhibitory effects on proliferation. Overall, these results demonstrate the development and application of age-specific cardiac CDMs to dissecting the influence of ECM proteins on cardiomyocyte function.

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