The Bone Morphogenetic Protein antagonist Gremlin 2 (Grem2) is required for atrial differentiation and establishment of cardiac rhythm during embryonic development. A human Grem2 variant has been associated with familial atrial fibrillation, suggesting that abnormal Grem2 activity causes arrhythmias. However, it is not known how Grem2 integrates into signaling pathways to direct atrial cardiomyocyte differentiation. Here, we demonstrate that Grem2 expression is induced concurrently with the emergence of cardiovascular progenitor cells during differentiation of mouse embryonic (ES) stem cells. Grem2 exposure enhances the cardiogenic potential of ES cells by ~20–120 fold, preferentially inducing genes expressed in atrial myocytes such as Myl7, Nppa and Sarcolipin. We show that Grem2 acts upstream to upregulate pro-atrial transcriptional factors CoupTFII and Hey1 and downregulate atrial fate repressors Irx4 and Hey2. The molecular phenotype of Grem2-induced atrial cardiomyocytes was further supported by induction of ion channels encoded by Kcnj3, Kcnj5, and Cacna1D genes and establishment of atrial-like action potentials shown by electrophysiological recordings. We show that promotion of atrial-like cardiomyocyte is specific to the Gremlin subfamily of BMP antagonists. Grem2 pro-atrial differentiation activity is conveyed by non-canonical BMP signaling through phosphorylation of JNK and can be reversed by specific JNK inhibitors, but not by dorsomorphin, an inhibitor of canonical BMP signaling. Taken together, our data provide novel mechanistic insights into atrial cardiomyocyte differentiation from pluripotent stem cells and will assist the development of future approaches to study and treat arrhythmias.
Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.
The well described Wnt inhibitor Dickkopf-1 (DKK1) plays a role in angiogenesis as well as regulation of growth factor signaling cascades in pulmonary remodeling associated with chronic lung diseases including emphysema and fibrosis. However, the specific mechanisms by which DKK1 influences mesenchymal vascular progenitor (MVPC), endothelial and smooth muscle cells within the microvascular niche have not been elucidated. In this study, we show that knock down of DKK1 in Abcg2pos lung mouse adult tissue resident MVPC alters lung stiffness, parenchymal collagen deposition, microvessel muscularization and density as well as loss of tissue structure in response to hypoxia exposure. To complement the in vivo mouse modeling, we also identified cell or disease specific responses to DKK1, in primary lung COPD MVPC, COPD MVEC and SMC, supporting a paradoxical disease specific response of cells to well-characterized factors. Cell responses to DKK1 were dose dependent and correlated with varying expression of the DKK1 receptor, CKAP4. These data demonstrate that DKK1 expression is necessary to maintain the microvascular niche while its effects are context specific. They also highlight DKK1 as a regulatory candidate to understand the role of Wnt and DKK1 signaling between cells of the microvascular niche during tissue homeostasis and during the development of chronic lung diseases.
Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents. Video LinkThe video component of this article can be found at
Background: End‐stage pulmonary arterial hypertension (PAH) is characterized by right ventricular (RV) dysfunction, failure and death. We have shown BMPR2 mutation is associated with impaired RV hypertrophy and cardiomyocyte lipid deposition in both heritable PAH patients and a murine model of mutant BMPR2 expression. Hypothesis: BMPR2 mutation alters hypertrophic response and fatty acid transport in RV cardiomyocytes. Methods: Stable mouse embryonic stem cell lines (ESC) were engineered to express BMPR2 mutation using pCI‐Neo‐BMPR2 plasmids with mutation in either cytoplasmic (CD) or kinase domain (KD) or a linearized pCI‐Neo vector (Control) and gene expression confirmed by RT‐PCR. Directed differentiation of the ESC to cardiac myocytes (ESC CM) performed using noggin. ESC CM treated with phenylephrine for 72 hours to induce hypertrophy. Western analysis performed for proteins: CD36 (a fatty acid transporter molecule), brain natriuretic peptide (BNP) and corin (a serine peptidase which cleaves pro‐BNP). Oil‐red‐O stain performed for intracellular lipid deposition in ESC‐CM. Results: All ESC‐CM expressed α‐SMA and demonstrated characteristic contraction in vitro. In response to hypertrophy, control ESC CM increased expression of BNP and corin, which remained unchanged in CD and KD BMPR2 mutant cells. At baseline expression of CD36 protein was higher in CD BMPR2 mutant containing ESC CM and Oil‐red‐O staining suggested increased intracellular lipid accumulation in mutant ESC CM relative to control cells. Hypertrophic stimulus reduced the expression of CD36 in CD and KD BMPR2 ESC CM compared to control cells. Conclusions: Altered BMPR2 signaling due to CD or KD mutations in ESC CM were associated with an impaired hypertrophic response. This impaired response was associated with decreased expression of BNP, corin and CD36. This may explain reduced ability of RV to adapt to increased RV afterload in PAH. Further characterization of the mechanism of impaired hypertrophy is planned. Grant Funding Source: P01HL108800
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