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Video protocol

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification
JoVE 3155 11/18/2011

May 2011 Detecting Autophagy in the Heart


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Detecting Autophagy in the Heart

C57BL/6-Tg(Myh6-mCherry/LC3)1Acvm RBRC03914

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Autophagosomes and autolysosomes in heart tissue of GFP/mCherry-LC3
double transgenic mice under starvation conditions

 

  Autophagy is an intracellular degradation process necessary for maintaining homeostasis. The process consists of two major parts; one is the transport of unfolded proteins and damaged organelles in double-membrane vesicles called autophagosomes, and another is the fusion of autophagosomes with lysosomes which contain hydrolytic enzymes for degrading their contents to form autolysosomes. The involvement of autophagy in heart diseases has been reported by using autophagy reporter mice that systemically express GFP-LC3 (RBRC00806) in autophagosomes. However, distinguishing autophagosomes and autolysosomes is required for detailed analysis of autophagic process under both normal and pathological conditions. To facilitate such analysis, new transgenic mice (C57BL/6-Tg(Myh6-mCherry/LC3)1Acvm), which express mCherry-LC3 by alphaMyHC promoter, were generated and crossed with GFP-LC3 transgenic mice. These double transgenic mice enable us to distinguish autophagosomes with GFP/mCherry and autolysosomes only with mCherry in cardiomyocytes. Because GFP is sensitive to the acidic conditions and attenuates in autolysosomes, while mCherry does not. Thus, the double transgenic GFP/mCherry-LC3 mice function as innovative and useful tools for examining the role of autophagy in the heart.

 

Depositor : Dr. Kiyoshi Nobori, Department of Cardiovascular Medicine, Akita University Graduate School of Medicine
Reference : Terada M, Nobori K, Munehisa Y, Kakizaki M, Ohba T, Takahashi Y, Koyama T, Terata Y, Ishida M, Iino K, Kosaka T, Watanabe H, Hasegawa H, Ito H. Double transgenic mice crossed GFP-LC3 transgenic mice with alphaMyHC-mCherry-LC3 transgenic mice are a new and useful tool to examine the role of autophagy in the heart. Circ J. 74(1):203-206, 2010.