The Expression of NPPA Splice Variants During Cardiac Differentiation of Mouse Mesenchymal and Embryonic Stem Cells Masoumeh Fakhr Taha PhD, Arash Javeri MD, PhD Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. Introduction: NPPA is an early and specific marker for functional myocardium of the embryonic heart. NPPA gene encodes for a precursor of atrial natriuretic peptide (ANP). The expression pattern of natriuretic peptide precursor A (NPPA) gene during development of cardiac chamber is highly dynamic. Moreover, the expression profile of NPPA gene is different between human and mouse. So far, three alternatively spliced variants have been reported for NPPA gene in human. In mouse, no alternatively spliced isoform of NPPA gene has been reported. In the current study, we studied the expression of NPPA gene during cardiac differentiation in vitro and in vivo. Materials and Methods: Cardiac differentiation of the ADSCs was induced by 5azacytidine, bone morphogenetic protein-4 (BMP4) or coculture with the mouse cardiomyocytes, and ES cells were differentiated at the presence or absence of bone morphogenetic protein-4 (BMP4). To assess the expression of NPPA splice variants during heart development, the cardiac area of 8-day mouse embryos and one-week old mice were isolated and used for evaluation. Left, Figure 3. (A) Location of the third set of NPPA primers spanning the exon-exon boundaries. (B) The expression of a 339 bp fragment which was amplified using the third set of NPPA primers (F3 and R3). Right, Figure 4. (A) The expression of alternatively spliced variants of NPPA in three-week differentiated ADSCs. (B) Sequencing of the d fragment revealed retention of the first 10 bp and terminal 142 bp of intron 2. (C) (I) The amino acid sequence of prepro-ANP. (II) Predicted peptide Figure 1. The expression of some cardiac-specific genes (Left) and proteins (Right) in two-week sequence for the alternatively spliced variants of NPPA mRNA. differentiated ADSCs of all the experimental groups. (A) The cells which were differentiated in 10% FBS-containing medium without any additional factor were used as the control group of differentiation. (B-E) Three-week differentiated ADSCs in the indirect coculture, direct coculture, 5-azacytidine and BMP4 groups, respectively. (F) The cardiomyocytes isolated from the hearts of one-week old mice were used as the positive control. Green color: Cardiac troponin-I immunostaining of the cardiomyocytes. Red color: Propodium iodide staining of the nuclei. Results: Two-week differentiated ADSCs and ES cells expressed some cardiacspecific makers, including atrial natriuretic peptide (ANP). Three additional intronretained splice variants of NPPA were also detected during cardiac differentiation of the ADSCs and ES cells. In addition, we detected three intron-retained splice variants of NPPA in 8-day mouse embryonic heart. In the mature cardiomyocytes of 1-week old mice, only the correctly spliced isoform of NPPA gene was expressed. Freshly isolated stromal vascular fraction also expressed one intron-retained isoform of NPPA gene. Figure 2. (A) Genomic organization of NPPA locus in mouse. Arrows marked as F2 and R2 indicate location of the second set of primers. (B) The expression of alternatively spliced variants of NPPA in the differentiated ADSCs, after three weeks and (C) after four weeks of cardiac induction. PCR was Figure 5. Cardiac differentiation of mouse ES cells. (A) Immunostaining of the ES cell-derived cardiomyocytes of the control and (B) BMP4 treated EBs for cardiac troponin-I protein. (C) The expression of two splice variants of NPPA mRNA in differentiated EBs of the control and BMP4 treatment groups. (D) The expression of NPPA splice variants in 8-day (E8) mouse embryonic heart. performed using the second set of primers. Neurogenic medium was used as the non-cardiogenic condition and mouse Conclusion: Our findings have provided evidence for the expression of intron- cardiomyocytes were used as the control. (D) The expression retained splices of NPPA mRNA during the early stages of mouse cardiogenesis as of one intron-retained isoform of NPPA in the freshly isolated well as in the mouse adipose tissue. However, the functional significance of these SVF cells. variants remains to be investigated.