CIC   05421
CENTRO DE INVESTIGACIONES CARDIOVASCULARES "DR. HORACIO EUGENIO CINGOLANI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Widespread reduction of sodium hydrogen exchanger isoform 1 across the heart after a single and local injection of siRNA in the mouse myocardium
Autor/es:
PE MORGAN, AA DIEZ, NG PEREZ, HE CINGOLANI
Lugar:
Berlin
Reunión:
Congreso; Frontiers in Cardiovascular Biology; 2010
Institución organizadora:
European Society of Cardiology
Resumen:
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Sodium hydrogen exchangers
belong to a family of ten members. NHE isoform 1 (SLC9A1) is the main isoform
expressed in the heart. Cardiac pathologies like ischemia/reperfusion injury
and hypertrophy have been associated to NHE1 hyperactivity, whose pharmacological
inhibition proved to be beneficial. RNA interference technology allows, specifically
and locally, to reduce protein expression. Small interference RNA was designed
against a 25 pb long mRNA target sequence specific for NHE1 (siRNANHE1).
The specificity was confirmed by NCBI BLAST tool. siRNA was produced in vitro by hybridization of complementary
single strand RNAs generated by a T7 RNA polymerase. As a negative control was
generated dsRNA with the scrambled target sequence (siRNASCR). siRNA
effectiveness was studied in HEK293 cells transfected with NHE1 cDNA alone or
co-transfected with either siRNANHE1 or siRNASCR. After
48 hs of transfection, HEK293 cells were lysated, electrophoresed and
inmunoblotted for NHE1 and GAPDH. HEK293 cells transfected with increasing
amounts of siRNANHE1 (mg): 0, 1, 4, 10, 40 reduced
NHE1 expression in a dose dependent manner and compared with siRNASCR
transfected cells; respectively: 100; 56.6 ± 3.5; 41.6 ± 6.8; 17.15 ± 0.2; 8.72
±7.4; 78.06 ± 3.1 (n=3 each, ANOVA One-way p<0.05). Thereafter, siRNA was
studied in vivo in the mouse heart. Anesthetised
mice had a small opening in the chest and the siRNA (40 mg) was injected site
at the apex in the myocardial wall of the left ventricle. Subsequently, the
chest was closed and 48/72 hs later the mice was sacrificed, hearts were excised
and analysed for protein expression on inmunoblots. NHE1 expression studied in
ventricle lysates showed a reduction for the siRNANHE1 treated mice compared
with the ones injected with siRNASCR (37.16 ± 5, n=5 and 93.7 ±
13.2, n=4; t-test p<0.05). Due to the extensive reduction of NHE1 expression
it was studied in three different parts of the left ventricle, namely apex,
medium body and base. NHE1 expression (reported as NHE1/GAPDH ratio) showed a
widespread reduction across the ventricle: Apex 0.126 ± 0.0331, n=3; Medium
0.225 ±
0.0788,
n=3; Base 0.343 ± 0.0984 n=3; siRNASCR 1.520 ± 0.1700, n=3 (ANOVA one way, P<0.05). In this report we
conclude that only one injection of naked siRNANHE1 in the
heart was enough to successfully reduce the NHE1 expression. A second and
remarkable finding is the ability of the siRNA molecule to achieve a reduction
of the NHE1 expression far away from its injection site. This finding corresponds
with a novel notion that siRNA molecules could diffuse through the heart syncytium.