Widespread reduction of sodium hydrogen exchanger isoform 1 across the heart after a single and local injection of siRNA in the mouse myocardium

PATRICIO E. MORGAN; ARIEL A. DIEZ; NÉSTOR G. PÉREZ; HORACIO E. CINGOLANI

Berlin

Congreso; Frontiers in Cardiovascular Biology, First Congress of the European Society of Cardiology Council on Basic Cardiovascular Science; 2010

European Society of Cardiology

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.