| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2009: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2008: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
|
|---|
| Research Abstract |
An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨ_m), is a crucial step in the cell death during myocardial ischemia/reperfusion ; reactive oxygen species (ROS) and Ca^<2+> are considered as the two major regulators of this step. We aimed to visualize the spatio-temporal relationship between ROS, Ca^<2+> and ΔΨ_m loss at cellular level in the perfused heart subjected to ischemia/reperfusion using a real-time two-photon imaging system. During ischemia/ reperfusion, ROS were mainly generated during early phase of ischemia with variable cell-to-cell specific rates and irreversible ΔΨ_m loss occurred in an all-or-none manner depending on cellular ROS level with a clear cut-off value. Cellular Ca^<2+> level concomitantly increased during ischemia, and once ROS level reached a threshold level, Ca^<2+> level increased robustly. Ischemic preconditioning attenuated both the increase of ROS and Ca^<2+> and protected a
… More
gainst ΔΨ_m loss. An anti-oxidant attenuated the ROS accumulation but not the Ca^<2+> increase ; it did not protect against ΔΨ_m loss. Thus, reducing only ROS accumulation does not suffice to prevent cardiomyocyte death ; suppression of both ROS and Ca^<2+> may be required.
An opening of the MPTP 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 aimed to investigate the protective effects of RNAi-mediated gene silencing of cyclophilin D (CypD), one of the putative components of the MPTP, against myocardial ischemia/reperfusion by using the two-photon laser scanning microscopy. We created an adenovirus carrying short interfering RNA (siRNA) which inactivates CypD. To investigate the effects in vivo, we monitored the spatio-temporal changes of ΔΨ_m in perfused rat hearts subjected to ischemia/reperfusion, using the two-photon imaging. Adult rats received direct intramyocardial injections of the adenovirus. Two to three days after injection, the rat hearts were perfused in Langendorff mode, and ΔΨ_m levels of individual cells were monitored. The progressive loss of ΔΨ_m during ischemia/reperfusion was significantly suppressed in CypD-siRNA-transduced cells, compared with non-transduced cells. Furthermore, the protective effect of CypD-siRNA was dose-dependent.
Thus, this novel two-photon imaging provides deeper insights into the mechanisms of cardiomyocyte death during ischemia/reperfusion, into cardioprotective therapy that targets mitochondria.. Less
|
