Early Postnatal Cardiac Changes and Premature Death in Transgenic Mice Overexpressing a Mutant Form of Serum Response Factor

Xiaomin Zhang,Jianyuan Chai,Gohar Azhar,Pamela Sheridan,Ana M Borras,Maxwell C Furr,Konstantin Khrapko,Joel Lawitts,Ravi P Misra,Jeanne Y Wei

doi:10.1074/jbc.m104934200

Abstract

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.

Highlights

Serum response factor (SRF)1 is a key regulator of many extracellular signal-regulated genes important for cell growth and differentiation [1,2,3]
The results showed that the binding activity of the dmSRF protein was much lower than that of the wild type SRF (wtSRF) protein (Fig. 1A)
To demonstrate that dmSRF can inhibit the binding of wtSRF to SRE in vitro, wtSRF protein was cotranslated with dmSRF

Summary

EXPERIMENTAL PROCEDURES

SRF Mutant—A functional double mutant form of human SRF gene, termed dmSRF, was generated by site-directed mutagenesis. In Vitro Translation and Cotranslation of wtSRF and dmSRF Protein—The DNA fragments corresponding to the full-length coding region of dmSRF and wild type SRF (wtSRF) were subcloned into plasmid pBluescript SK(-). The transcription of both the mutant and wild type SRF genes was under the control of T7 promoter. Gel supershift assays were performed as described above with the exception that subsequent to incubation of oligonucleotide probes with the whole cell extract, 1 ␮l of of anti-SRF antibody (1 ␮g/␮l; Santa Cruz Biotechnology) was added to the reaction mixture and incubated at room temperature for 30 min. The measurements from six electron micrographs from each specimen were averaged for each LV region in each group

Transgenic progeny

RESULTS

DISCUSSION