Chapter 16 Novel Methods for Measuring Cardiac Autophagy In Vivo

Cynthia N. Perry, Shiori Kyoi, Nirmala Hariharan, Hiromitsu Takagi, Junichi Sadoshima, Roberta A. Gottlieb

Research output: Contribution to journalReview article

47 Scopus citations

Abstract

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, occurs constitutively in the heart and may serve as a cardioprotective mechanism in some situations. It has been implicated in the development of heart failure and is up-regulated following ischemia-reperfusion injury. Autophagic flux, a measure of autophagic vesicle formation and clearance, is an important measurement in evaluating the efficacy of the pathway, however, tools to measure flux in vivo have been limited. Here, we describe the use of monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine to measure autophagic flux in in vivo model systems, specifically focusing on its use in the myocardium. This method allows determination of flux as a more precise measure of autophagic activity in vivo much in the same way that Bafilomycin A1 is used to measure flux in cell culture. MDC injected 1 h before sacrifice, colocalizes with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This chapter provides a method to measure autophagic flux in vivo in both transgenic and nontransgenic animals, using MDC and chloroquine, and in addition describes the mCherry-LC3 mouse and the advantages of this animal model in the study of cardiac autophagy. Additionally, we review several methods for inducing autophagy in the myocardium under pathological conditions such as myocardial infarction, ischemia/ reperfusion, pressure overloading, and nutrient starvation.

Original languageEnglish (US)
Pages (from-to)325-342
Number of pages18
JournalMethods in Enzymology
Volume453
Issue numberC
DOIs
StatePublished - Feb 12 2009
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Perry, C. N., Kyoi, S., Hariharan, N., Takagi, H., Sadoshima, J., & Gottlieb, R. A. (2009). Chapter 16 Novel Methods for Measuring Cardiac Autophagy In Vivo. Methods in Enzymology, 453(C), 325-342. https://doi.org/10.1016/S0076-6879(08)04016-0