The Bone Morphogenetic Protein antagonist Gremlin 2 (Grem2) is required for atrial differentiation and establishment of cardiac rhythm during embryonic development. Gremlin subfamily of BMP antagonists. Grem2 pro-atrial differentiation activity is conveyed by non-canonical BMP signaling through phosphorylation of JNK and IL17RA antibody 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 Amonafide (AS1413) will assist the development of future approaches to study and treat arrhythmias. INTRODUCTION Embryonic stem (ES) cells differentiate to a wide range of cell types offering a robust system to obtain cells to study developmental mechanisms and disease phenotypes [1 2 The ES cell model is particularly pertinent for generating cells of the cardiovascular system because these cells appear relatively early during development and ES cell differentiation [3-7]. A number of experimental protocols exist to promote the differentiation of ES cells toward cardiac cell fates [8-15]; however how to direct ES cell-derived cardiac progenitors to cultures of specialized cell types such as ventricular and atrial myocytes pacemaker and conduction system cells remains a major challenge [16]. Bone Morphogenetic Proteins (BMPs) exert pleiotropic effects on cardiac morphogenesis and cardiomyocyte maturation [17] including cardiac looping [18 19 valve formation and ventricular development [20-26]. Besides forward BMP signaling BMP antagonists such as Amonafide (AS1413) Noggin are also necessary for cardiac development. Mice lacking Noggin have Amonafide (AS1413) thicker myocardium than wild types [27]. This phenotype could be rescued by halving the gene dosage of expression has been detected in commissural neurons of the developing spinal cord and in lung mesenchyme [33 34 studies in animal models have implicated Grem2 in Amonafide (AS1413) follicle development placode neurogenesis osteogenic differentiation and craniofacial patterning [32 35 Our prior studies have shown that Grem2 is usually highly expressed in the eye swim bladder and in the pharyngeal arch mesoderm adjacent to the developing heart of zebrafish embryos [38]. We decided that through regulation of BMP signaling Grem2 is necessary for cardiac laterality and atrial differentiation during development [39]. In addition we discovered that a human variant is associated with familial atrial fibrillation suggesting that abnormal Grem2 activity causes arrhythmia. Modeling of the human variant resulted in slower cardiac contraction rates abnormal atrial contraction velocity and distorted wavefront propagation in zebrafish supporting the idea that Grem2 regulates the establishment of proper cardiac rhythm in the atrium. Furthermore we found that Grem2 overexpression during development led to ectopic contracting fields expressing atrial-specific genes; thus Grem2 activity is necessary and sufficient for atrial differentiation [39]. Here we show that Grem2 treatment shifts ES cell differentiation to cardiomyocytes with atrial molecular and electrophysiological properties. This Grem2 effect is driven by activation of the JNK signaling pathway. Our findings Amonafide (AS1413) provide novel mechanistic insights into chamber-specific cardiomyocyte differentiation and the development of stem cell-based tools to study and treat atrial dysfunction. MATERIALS AND METHODS ES cell culture and embryoid body (EB) formation Mouse CGR8 ES cells have been adapted to feeder-free culture conditions facilitating molecular analyses of gene expression [7 14 39 CGR8 cells were cultured in GMEM medium (Sigma) with 10% fetal bovine serum 100 units/ml LIF (ESGRO-Millipore) 2 mM L-glutamine and 50 μM β-mercaptoethanol on 0.2% gelatin coated tissue culture plates. Ha sido cells were held at ≤ 70% confluence to protect pluripotency. Ha sido cell differentiation was performed using the hanging-drop technique [14 42 Quickly ES cells had been dissociated with 0.05% trypsin (Life Technologies) for five minutes and 500 cells in 20 μl drops were Amonafide (AS1413) spotted inside lid surface of the 10 cm bacterial Petri dish. EBs had been harvested in differentiation moderate [IMDM (Lifestyle Technologies) formulated with 20% FBS 0.1 mM MEM important proteins 2 mM-glutamine and 100 μM.