Conditional dicer gene depletion in the postnatal myocardium

Conditional Dicer Gene
Depletion in the
Postnatal Myocardium
Provokes Spontaneous
Cardiac Remodeling
MARTINS, P. A. C.; BOURAJJAJ, M.; GLADKA, M.;
KORTLAND, M.; OORT,R. J. V.; PINTO, Y. M.;
MOLKENTIN, J. D.; WINDT, L. J.

Circulation, v. 118, n. 15, oct. 2008
American Heart Association

Aluna: Dayana P. P. Siqueira

INTRODUCTION miRNA Endogenous
Regulate gene expression Small noncoding Drosha
Dicer

Mature miRNA
Pri-miRNA

(18 to 24 nucleotides long)

AMBROS, 2003; FARH, et al., 2005; PASQUINELLI, HUNTER, BRACHT, 2005; CULLEN, 2004.

RNA-induced silencing complex

BARTEL, 2004; BENTWICH et al., 2005; BEREZIKOV et al., 2005.

Negatively regulate protein expression by either mRNA degradation or translational inhibition

miRNA

May regulate up 1/3 of the human genome

Current studies

Multiple transcripts

Fundamental role in regulating gene expression OBJECTIVES

Trigger loss of Dicer in the postnatal myocardium through the use of a tamoxifen-inducible Cre recombinase expressed in murine heart cells.

METHODS
Transgenic Mice

Cre

Mice were treated with:
• vehicle or tamoxifen (20mg/Kg per day)
• intraperitoneal injections for consecutive 5 days

3 weeks

8 weeks

Western Blotting
Proteins were extracted from mouse cardiac tissue

Antibodies against:

Dicer (rabbit polyclonal)
GAPDH (mouse monoclonal)

Irx5 (mouse monoclonal)
Kcnd2 (rabbit polyclonal)

Histological Analysis and Immunofluorescence Microscopy

Hearts were arrested in diastole
Perfusion-fixed with 4% paraformaldehyde
Embedded in parafin

Sectioned at 5µm
H&E

routine histological analysis

Sirius red

detection of fibrillar collagen

WGA

visualize and quantify the cross-sectional area of cardiomyocytes CD31

visualize and quantify the number of capillaries per myofiber Transthoracic Echocardiography

Were performed on mice anesthetized with isoflurane
1 week and 4 weeks