2010 Fiscal Year Final Research Report
Two-photon laser scanning microscopy for the investigation of cardiomyocyte death during ischemia/reperfusion.
| Project/Area Number |
20590862
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Kyoto University |
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Principal Investigator |
AKAO Masaharu Kyoto University, 展開医療部, 主任研究員 (00362509)
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| Project Period (FY) |
2008 – 2010
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| Keywords | 分子心臓病態学 |
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| 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
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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
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Research Products
(12 results)
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[Journal Article] p300 plays a critical role in maintaining cardiac mitochondrial function and cell survival in postnatal hearts.2009
Author(s)
Nakagawa Y, Kuwahara K, Takemura G, Akao M, Kato M, Arai Y, Takano M, Harada M, Murakami M, Nakanishi M, Usami S, Yasuno S, Kinoshita H, Fujiwara M, Ueshima K, Nakao K.
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Journal Title
Circ Res. 105(8)
Pages: 746-54
Peer Reviewed
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