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.

Conclusion: Our findings have provided evidence for the expression of intron-

retained splices of NPPA mRNA during the early stages of mouse cardiogenesis as

well as in the mouse adipose tissue. However, the functional significance of these

variants remains to be investigated.

Results: Two-week differentiated ADSCs and ES cells expressed some cardiac-

specific makers, including atrial natriuretic peptide (ANP). Three additional intron-

retained 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 1. The expression of some cardiac-specific genes (Left) and proteins (Right) in two-week

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.

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

performed using the second set of primers. Neurogenic

medium was used as the non-cardiogenic condition and mouse

cardiomyocytes were used as the control. (D) The expression

of one intron-retained isoform of NPPA in the freshly isolated

SVF cells.

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

sequence for the alternatively spliced variants of NPPA mRNA.

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.

Materials and Methods: Cardiac differentiation of the ADSCs was induced by 5-

azacytidine, 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.