Autophagy is an intracellular process in which proteins and organelles are transported in double-membrane vesicles called autophagosomes through the cytoplasm to lysosomes for degradation. The autophagosome acquires hydrolytic enzymes by fusing with the lysosome to generate an autolysosome. Constitutive autophagy in the heart under baseline conditions is a homeostatic mechanism for maintaining cardiomyocyte size and global cardiac structure and function. Upregulation of autophagy in various heart diseases, including cardiac hypertrophy and heart failure, is an adaptive response for protecting cells from hemodynamic stress. However, the detailed roles of autophagy in the heart remain unclear. LC3 is localized on the autophagosome membrane. Exogenously expressed GFP fused to LC3 (GFP-LC3) serves as an ideal molecular marker for autophagosome. Transgenic mice expressing GFP-LC3 (CAG-GFP-LC3) have been used to detect autophagy systemically. However, CAG-GFP-LC3 mice cannot distinguish autophagy-positive cardiomyocytes from other cells such as fibroblasts and smooth muscles in the heart and cannot detect autolysosome because GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions. To resolve these problems, we have generated transgenic mice (αMyHC-mCherry-LC3) expressing mCherry fused to LC3 under the control of αmyosin heavy chain promoter instead of CAG promoter to detect autophagy only in cardiomyocytes. mCherry is an improved-monomeric red-fluorescence protein and does not lose fluorescence under acidic condition. Thus, αMyHC-mCherry-LC3 mice can detect not only autophagosome before fusion with lysosome but also autophagosome after fusion with lysosome. Moreover, we have crossed αMyHC-mCherry-LC3 mice with CAG-GFP-LC3 mice. Green signals showed autophagosome in non-cardiomyocytes. On the other hand, red signals showed autolysosome and double positive signals showed autophagosome in cardiacmyocytes. In conclusion, we have generated αMyHC-mCherry-LC3 mice to detect both autophagosome and autolysosome. The double transgenic mice cannot only detect autophagosome and autolysosome but also distinguish between them. This is an innovative method to examine the role of autophagy in cardiomyocytes.
Effect of Hypertriglyceridemia on Beta Cell Mass and Function in ApoC3 Transgenic Mice