Biology Reference
In-Depth Information
Abstract
Cellular energy homeostasis is a crucial function of oxidative tissues but becomes
altered with obesity, a major health problem that is rising unabated and demands
attention. Maintaining cardiac lipid homeostasis relies on complex processes and
pathways that require concerted actions between lipid droplets (LDs) and mitochon-
dria to prevent intracellular accumulation of bioactive or toxic lipids while providing
an efficient supply of lipid for conversion into ATP. While cardiac mitochondria
have been extensively studied, cardiac LDs and their role in heart function have
not been fully characterized. The cardiac LD compartment is highly dynamic and
individual LD is small, making their study challenging. Here, we describe a simple
procedure to isolate cardiac LDs that provide sufficient amounts of highly enriched
material to allow subsequent protein and lipid biochemical characterization. We also
present a detailed protocol to image cardiac LDs by conventional transmission
electronic microscopy to provide two-dimensional (2D) analyses of cardiac LDs
and mitochondria. Finally, we discuss the potential advantages of dual ion beam
and electron beam platform (FIB-SEM) technology to study the cardiac LDs and
mitochondria by allowing 3D imaging analysis.
INTRODUCTION AND RATIONALE
Maintaining appropriate cellular lipid homeostasis is crucial to heart function, and
numerous studies have established that lipid homeostasis is compromised in
obesity-induced cardiomyopathy, a rapidly rising health problem (
Cornier et al.,
2008; Lopaschuk, Ussher, Folmes, Jaswal, & Stanley, 2010; Szczepaniak, Victor,
Orci, & Unger, 2007
). To better understand molecular underpinnings of cardiomy-
opathy and develop targeted preventive and therapeutic treatments, we need to iden-
tify the pathways and genes regulating cardiac cellular lipid homeostasis. Distinct
among features of cardiomyopathy in obese and diabetic patients are association
of increased incidence of heart failure with the accumulation of cardiomyocyte lipid
droplets (LDs) (
Borradaile & Schaffer, 2005; Khan, Drosatos, & Goldberg, 2010;
Lopaschuk et al., 2010; Wende & Abel, 2010
). Ample evidence indicates that
LDs play a critical, cardioprotective role in lipid homeostasis by mediating transient
storage of excess fatty acid (FA) in the form of triglycerides (
Listenberger et al.,
2003; Liu et al., 2007
). Cardioprotection by LD from accumulation of bioactive
lipids or production of toxic lipid species occurs at times when FA supply exceeds
demand, for example, fasting, as demonstrated in both rodent and human heart tissue
(
Jacob, 1987; Reingold et al., 2005
). However, role of LDs in cardiomyopathy re-
mains underappreciated and controversial (
Brindley, Kok, Kienesberger, Lehner,
& Dyck, 2010; Park, Yamashita, Blaner, & Goldberg, 2007
). Excess fat in cardi-
omyocytes is associated with cardiac lipotoxicity/dysfunction in mice models
with specific heart overexpression or downregulation of genes involved in lipid
