Michael Trembley

Myocardin-related transcription factors control the motility of epicardium-derived cells and the maturation of coronary vessels

An important pool of cardiovascular progenitor cells arises from the epicardium, a single layer of mesothelium lining the heart. Epicardium-derived progenitor cell (EPDC) formation requires epithelial-to-mesenchymal transition (EMT) and the subsequent migration of these cells into the sub-epicardial space. Although some of the physiological signals that promote EMT are understood, the functional mediators of EPDC motility and differentiation are not known. Here, we identify a novel regulatory mechanism of EPDC mobilization. Myocardin-related transcription factor (MRTF)-A and MRTF-B (MKL1 and MKL2, respectively) are enriched in the perinuclear space of epicardial cells during development. Transforming growth factor (TGF)-β signaling and disassembly of cell contacts leads to nuclear accumulation of MRTFs and the activation of the motile gene expression program. Conditional ablation of Mrtfa and Mrtfb specifically in the epicardium disrupts cell migration and leads to sub-epicardial hemorrhage, partially stemming from the depletion of coronary pericytes. Using lineage-tracing analyses, we demonstrate that sub-epicardial pericytes arise from EPDCs in a process that requires the MRTF-dependent motile gene expression program. These findings provide novel mechanisms linking EPDC motility and differentiation, shed light on the transcriptional control of coronary microvascular maturation and suggest novel therapeutic strategies to manipulate epicardium-derived progenitor cells for cardiac repair. Highlighted article: Myocardin-related transcription factors respond to TGFβ signalling and control both the motility of mouse epicardium-derived progenitors and the maturation of coronary vessels.

Tension Creates an Endoreplication Wavefront that Leads Regeneration of Epicardial Tissue

Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.

Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells.

Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Mechanosensitive Gene Regulation by Myocardin-Related Transcription Factors is Required for Cardiomyocyte Integrity in Load-Induced Ventricular Hypertrophy

Background: Hypertrophic cardiomyocyte growth and dysfunction accompany various forms of heart disease. The mechanisms responsible for transcriptional changes that affect cardiac physiology and the transition to heart failure are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling cardiomyocyte force transmission and propagation of electrical activity. The ID is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. Methods: Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor (MRTF)-A and MRTF-B specifically in adult cardiomyocytes to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. Results: We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure. Although mice lacking MRTFs in adult cardiomyocytes display normal cardiac physiology at baseline, pressure overload leads to rapid heart failure characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and cardiomyocyte adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by single-molecule localization microscopy may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. Conclusions: Our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates cross-talk between the actin and microtubule cytoskeleton and maintains ID integrity and cardiomyocyte homeostasis in heart disease.