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 language | English (US) |
|---|---|
| Pages (from-to) | 325-342 |
| Number of pages | 18 |
| Journal | Methods in Enzymology |
| Volume | 453 |
| Issue number | C |
| DOIs | |
| State | Published - Feb 12 2009 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
Cite this
Chapter 16 Novel Methods for Measuring Cardiac Autophagy In Vivo. / Perry, Cynthia N.; Kyoi, Shiori; Hariharan, Nirmala; Takagi, Hiromitsu; Sadoshima, Junichi; Gottlieb, Roberta A.
In: Methods in Enzymology, Vol. 453, No. C, 12.02.2009, p. 325-342.Research output: Contribution to journal › Review article
}
TY - JOUR
T1 - Chapter 16 Novel Methods for Measuring Cardiac Autophagy In Vivo
AU - Perry, Cynthia N.
AU - Kyoi, Shiori
AU - Hariharan, Nirmala
AU - Takagi, Hiromitsu
AU - Sadoshima, Junichi
AU - Gottlieb, Roberta A.
PY - 2009/2/12
Y1 - 2009/2/12
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=59649084638&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(08)04016-0
DO - 10.1016/S0076-6879(08)04016-0
M3 - Review article
C2 - 19216914
AN - SCOPUS:59649084638
VL - 453
SP - 325
EP - 342
JO - Methods
JF - Methods
SN - 0076-6879
IS - C
ER -