Heart Protection Using a New Approach - Antisense to Angiotensin Converting Enzyme Mrna
Introduction. Angiotensin converting enzyme (ACE), plays a major role in many cardiovascular pathophysiological conditions, such as hypertension, heart failure and myocardial ischemia. Currently used ACE inhibitors have some adverse effects and non-compliance problem. Therefore, our lab has been developing an antisense approach to inhibit the renin-angiotensin system at the gene expression level. The present study had three aims: 1. Design antisense oligodeoxynucleotide (AS-ODN) directed at ACE mRNA 2. Check tissue distribution of AS after iv injection 3. Test AS for heart protection in ischemia- reperfusion injury Methods. Epifluorescent and confocal laser-scanning microscopy, immunohistochemistry, Western blotting. Animals : mice, males, 20 g and Sprague-Dawley rats, males, 200-250 g. AS 5’ -ATTTCGTGGTGGG-3’ , inverted control (INV) 5’ -GGGTGGTGCTTTA-3’, phosphorothioates mixed with liposomes and injected iv 0.04 mg/mouse and 0.2 mg/rat. Captopril dose was 5 mg/kg. Isolated perfused heart function recordings : coronary perfusion pressure (CPP), left ventricular end-diastolic (LVEDP) and systolic pressures (LVSP). Results. Based on the rat ACE cDNA sequence, the AS was designed as 13-mer directed to the second zinc-binding domain (2998-3010 bp). Tissue distribution of phosphorothioated, fluorescein isothiocyanate (FITC)-labeled AS, 4h after iv injection, showed its presence in the lung, spleen, liver and heart, but absence in the brain. Immunostaining of the parallel heart slices with anti sarcomeric alpha-actinin antibody indicated AS localization within cardiomyocytes. ACE AS injected iv to the rats 24 h before excising the heart, which was then subjected to 25 min ischemia and 30 min reperfusion, preserved heart function. Increase in CPP and LVEDP during reperfusion was attenuated by AS as compared to INV or saline treatment (n=8 in each group, p<0.05). Moreover, ACE protein was not upregulated, as it was in the captopril-treated group. Conclusion. ACE AS is taken up by the heart tissue and provides cardioprotection by effectively preventing myocardial dysfunction caused by ischemia-reperfusion injury.