REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects.

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.

Hey2 regulates the size of the cardiac progenitor pool during vertebrate heart development.

A key event in heart development is the timely addition of cardiac progenitor cells, defects in which can lead to congenital heart defects. However, how the balance and proportion of progenitor proliferation versus addition to the heart is regulated remains poorly understood. Here, we demonstrate that Hey2 functions to regulate the dynamics of cardiac progenitor addition to the zebrafish heart. We found that the previously noted increase in myocardial cell number found in the absence of Hey2 function was due to a pronounced expansion in the size of the cardiac progenitor pool. Expression analysis and lineage tracing of hey2-expressing cells showed that hey2 is active in cardiac progenitors. Hey2 acted to limit proliferation of cardiac progenitors, prior to heart tube formation. Use of a transplantation approach demonstrated a likely cell-autonomous (in cardiac progenitors) function for Hey2. Taken together, our data suggest a previously unappreciated role for Hey2 in controlling the proliferative capacity of cardiac progenitors, affecting the subsequent contribution of late-differentiating cardiac progenitors to the developing vertebrate heart.

sfrp1 promotes cardiomyocyte differentiation in Xenopus via negative-feedback regulation of Wnt signalling.

Wnt signalling is a key regulator of vertebrate heart development, yet it is unclear which specific Wnt signalling components are required to regulate which aspect of cardiogenesis. Previously, we identified Wnt6 as an endogenous Wnt ligand required for controlling heart muscle differentiation via canonical Wnt/ß-catenin signalling. Here we show for the first time a requirement for an endogenous Wnt signalling inhibitor for normal heart muscle differentiation. Expression of sfrp1 is strongly induced in differentiating heart muscle. We show that sfrp1 is not only able to promote heart muscle differentiation but is also required for the formation of normal size heart muscle in the embryo. sfrp1 is functionally able to inhibit Wnt6 signalling and its requirement during heart development relates to relieving the cardiogenesis-restricting function of endogenous wnt6. In turn, we discover that sfrp1 expression in the heart is regulated by Wnt6 signalling, which for the first time indicates that sfrp genes can function as part of a Wnt negative-feedback regulatory loop. Our experiments indicate that sfrp1 controls the size of the differentiating heart muscle primarily by regulating cell fate within the cardiac mesoderm between muscular and non-muscular cell lineages. The cardiac mesoderm is therefore not passively patterned by signals from the surrounding tissue, but regulates its differentiation into muscular and non-muscular tissue using positional information from the surrounding tissue. This regulatory network might ensure that Wnt activation enables expansion and migration of cardiac progenitors, followed by Wnt inhibition permitting cardiomyocyte differentiation.