Abstract 1490: The Targeting Of Cyclophilin D By RNA Interference As A Novel Therapeutic Strategy Against Myocardial Ischemia/Reperfusion Injury
Background- An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨm) and mitochondrial dysfunction, is the earliest event that commits cells to death, and this process is potentially a prime target for therapeutic intervention against myocardial ischemia/reperfusion. We examined the effects of genetic knock-down of cyclophilin D (CypD), one of the putative components of MPTP, using RNA interference.
Methods and Results- We created an adenovirus carrying short interfering RNA (siRNA) which inactivates CypD. Transduction of siRNA-CypD in cultured neonatal rat cardiomyocytes achieved 68% reduction in mRNA and 61% reduction in protein levels, and suppressed cell death induced by H2O2 (100μM) as assessed by cell viability assay. It also protected against ΔΨm loss, as assessed by both time-lapse confocal microscopy and fluorescence-activated cell sorter analysis of cardiomyocytes loaded with an indicator of ΔΨm, tetramethylrhodamine ethylester (TMRE). To further investigate the effects in vivo, we monitored the spatio-temporal changes of ΔΨm in perfused rat hearts subjected to ischemia/reperfusion, using the real-time two-photon confocal imaging system which we recently developed. Briefly, adult rats received direct intramyocardial injections of the siRNA-CypD adenovirus. 3–5 days after injection, the rat hearts were removed and perfused in Langendorff mode. After loading TMRE, the hearts underwent two-photon imaging under excitation with 810 nm line of a Ti:Sapphire laser. Ischemia was achieved by clamping the perfusion line, and reperfusion was achieved by releasing the clamp. The virus contained an expression cassette of green fluorescent protein, allowing us to distinguish transduced cells and non-transduced cells. The progressive loss of ΔΨm induced by myocardial ischemia/ reperfusion was significantly suppressed in siRNA-CypD-transduced cells, compared with non-transduced cells.
Conclusions- Targeting CypD by RNA interference protects against oxidant-induced cardiomyocyte injury in vitro, and myocardial ischemia/reperfusion in vivo, implicating CypD as a promising molecular target for anti-ischemia/reperfusion therapy.