Evaluating mitochondrial autophagy in the mouse heart.

Area of Science:

• Cell Biology
• Molecular Biology
• Physiology

Background:

• Mitochondrial autophagy is crucial for maintaining mitochondrial quality control.
• Assessing mitochondrial autophagy in adult hearts in vivo presents significant challenges.
• Mito-Keima, a pH-sensitive fluorescent protein, has shown promise for evaluating mitochondrial autophagy in vitro.

Purpose of the Study:

• To develop and validate a method for evaluating mitochondrial autophagy in the adult heart in vivo.
• To investigate the impact of fasting on mitochondrial autophagy in the heart.

Main Methods:

• Generation of adeno-associated virus (AAV) serotype 9 vectors encoding Mito-Keima and Lamp1-YFP.
• Intravenous administration of AAV vectors into mice, followed by a 48-hour fasting or normal chow regimen.
• Confocal and electron microscopic analyses of heart tissue to assess mitochondrial autophagy and lysosomal co-localization.

Main Results:

• Mito-Keima puncta co-localized significantly with Lamp1-YFP, confirming lysosomal localization.
• Fasting significantly increased the co-localization of Mito-Keima and Lamp1-YFP, indicating heightened mitochondrial autophagy.
• Electron microscopy revealed mitochondrial autophagosomes post-fasting, and mitochondrial mass indicators (mtDNA, COX1/GAPDH) were reduced.

Conclusions:

• Intravenous injection of AAV-Mito-Keima and AAV-Lamp1-YFP enables in vivo evaluation of mitochondrial autophagy in the adult heart.
• Fasting stimulates lysosomal degradation of mitochondria in the heart, enhancing mitochondrial quality control.