Biology Reference
In-Depth Information
Wagenmaker, 2008
). In skeletal muscle cells where lipids are utilized for energy,
interaction between LDs and mitochondria is enhanced by exercise (
Tarnopolsky
et al., 2007
). More recently, it was shown that perilipin 5, a LD-associated protein,
stabilizes LDs in close proximity to mitochondria (
Bosma et al., 2012; Wang et al.,
2011
). Therefore, continuing work is required to elucidate mechanisms underlying
physical and metabolic relationships between cardiac LDs and mitochondria as well
as its physiological and pathological relevance.
Development of robust techniques is important to analyze the cardiac LD-
associated proteins and the spatial organization of the network of mitochondria
and LDs from heart tissues; determination of content and distribution could provide
crucial information. Major limitations to study cardiac LDs are their relative small
size and low abundance in the heart. While the remarkable lipid storage capacity of
white adipocyte LDs can be readily visualized by microscopic observation, cardiac
LDs are best visualized by electron microscopy under the most physiological con-
ditions possible. Mature adipocytes have a single LD whose size can range from
25 to 150
m diameter occupying most of the cell volume; steatotic liver can contain
LD with a size reaching 10
m
m
m, while cardiac LD size usually averages less than
m
1
m in diameter and are always in close association to mitochondria. Here, we pro-
vide detailed protocols that are the basis for our published methods for isolating car-
diac LDs and for analyzing cardiac LD proteins by western blot. Also included is a
protocol we have used to image cardiac LDs by conventional transmission electronic
microscope. In addition, we discuss the potentiality of novel three-dimensional (3D)
methodologies for imaging and quantifying cardiac LD and mitochondrial size using
Dual beam FIB-SEM.
8.1
ISOLATION OF CARDIAC LD PROTEINS AND PREPARATION
OF CARDIAC LDs FOR ANALYSIS BY WESTERN BLOT
There are many published techniques to isolate LDs from cells and tissues of
different organisms (
Brasaemle & Wolins, 2006; Ding et al., 2013; Harris, Shew,
Skinner, & Wolins, 2012
). All these techniques rely on fractionation by differential
centrifugation, benefiting from the relative low density of these organelles that
makes them float in aqueous solution. A major and common caveat of LD fraction-
ation is the remaining level of unwanted membranes/proteins from other cellular or-
ganelles in the low density LD fraction, especially from ER and mitochondria.
Conditions used for cell lysis or tissue homogenization are likely to be the main fac-
tor determining the levels of these unwanted contaminants. Attempts to remediate
have commonly included additional washes at similar or increased pH (carbonate
wash buffer at pH 10). However, these additional steps run a risk for loss of the
scarce amounts of extractable LDs from the heart. Cardiac LDs are in low abundance
and are very labile upon nutritional conditions. For example, they are almost unde-
tectable in mice studied in fed conditions, but their presence increases with increas-
ing time of fasting (
Pollak et al., 2013; Wang et al., 2013
). In addition, the abundance
