The targeting of cyclophilin D by RNAi as a novel cardioprotective therapy: evidence from two-photon imaging.
OBJECTIVE
An opening of the mitochondrial permeability transition pore (MPTP), which leads to the loss of mitochondrial membrane potential (DeltaPsi(m)), is the earliest event that commits cells to death, and this process is potentially a prime target for therapeutic intervention against myocardial ischaemia/reperfusion. We aimed to investigate the protective effects of RNA interference (RNAi)-mediated gene silencing of cyclophilin D (CypD), one of the putative components of the MPTP, against myocardial ischaemia/reperfusion using two-photon laser scanning microscopy.


RESULTS
We created an adenovirus carrying short-interfering RNA (siRNA) that inactivates CypD. Transduction of CypD-siRNA in rat cardiomyocytes achieved a 61% reduction in CypD mRNA and a 63% reduction in protein levels as well as protection against oxidant-induced DeltaPsi(m) loss and cytotoxicity. To further investigate the effects in vivo, we monitored the spatio-temporal changes of DeltaPsi(m) in perfused rat hearts subjected to ischaemia/reperfusion using two-photon imaging. Adult rats received direct intramyocardial injections of the adenovirus. Two to three days after injection, rat hearts were perfused by the Langendorff method and DeltaPsi(m) levels of individual cells were monitored. The progressive loss of DeltaPsi(m) during ischaemia/reperfusion was significantly suppressed in CypD-siRNA-transduced cells compared with non-transduced cells. Furthermore, the protective effect of CypD-siRNA was dose-dependent.


CONCLUSIONS
Therapeutic interventions designed to inactivate CypD may be a promising strategy for reducing cardiac injury against myocardial ischaemia/reperfusion. The two-photon imaging technique provides deeper insight into cardioprotective therapy that targets mitochondria.
Kato
Masashi
M
Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Akao
Masaharu
M
Matsumoto-Ida
Madoka
M
Makiyama
Takeru
T
Iguchi
Moritake
M
Takeda
Toshihiro
T
Shimizu
Shigeomi
S
Kita
Toru
T
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism*; D019610:Cytoprotection; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D008297:Male; D053078:Membrane Potential, Mitochondrial; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000473:pathology; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000201:enzymology* / Q000473:pathology; D018384:Oxidative Stress; D010477:Perfusion; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014161:Transduction, Genetic
D000081406:Peptidyl-Prolyl Isomerase F; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D021983:Cyclophilins