From the Gladstone Institute of Cardiovascular Disease (L.B.N., M.V.,
J.B., M.R., L.F., S.G.Y.), Cardiovascular Research Institute (L.B.N., M.V.,
J.B., M.R., S.G.Y., M.D.G., R.L.H., J.S.W., C.T.), and Departments of Medicine
(S.G.Y., M.D.G.) and Anatomy (R.L.H., J.S.W.), University of California, San
Francisco; and the Department of Medicine, Columbia University College of
Physicians and Surgeons, New York, NY (I.J.G., T.V.-R.).
Methods and Results—Northern blot and RNase protection
analyses showed that the apoB and MTP genes were expressed in
the hearts of mice and humans. In situ hybridization studies revealed
that the apoB mRNA was produced in cardiac myocytes. Electron
microscopy of human cardiac myocytes revealed lipid-staining particles
of relatively small diameter (
Conclusions—These studies strongly suggest that the
heart synthesizes and secretes apoB-containing lipoproteins.
Our laboratory recently used an 80-kb P1 bacteriophage clone
(p158) spanning the entire human apoB gene to generate human apoB
transgenic mice.3 When RNA samples from various
transgenic mouse tissues were analyzed for transgene
expression, we made two unexpected findings. First, although the
transgene was highly expressed in the liver, transgene expression was
absent in the intestine. Second, the human apoB transgene was expressed
in the heart, an organ thought to lack apoB gene expression.
Quantification of the RNA slot blots revealed that apoB gene expression
in the heart was at significant levels, 4% of that in the liver, and
easily detectable after a short exposure of the RNA slot
blot.3 Heart apoB expression was also noted by
Callow et al,4 who also used p158 to generate
human apoB transgenic mice.
In interpreting these results, we hypothesized that the peculiar
pattern of transgene expression arose because p158 was simply too short
to contain the cis-acting DNA sequence elements governing
the "correct" spatial pattern of apoB gene expression. Our first
assumption was that the human apoB transgene lacked an enhancer element
governing apoB gene expression in the intestine. This assumption proved
to be correct; subsequent studies revealed that the intestinal
expression of the apoB gene is governed by a distant intestinal
enhancer element located more than 30 kb upstream from the apoB
gene.5 6
In the present study, we examined whether human apoB
expression in the hearts of the human apoB transgenic mice was a
"transgenic artifact" or whether the expression of apoB in the
heart might reflect an unrecognized capacity of the heart to synthesize
lipoproteins. Our studies revealed that the expression of apoB in the
heart is a normal feature of cardiac gene expression. Interestingly,
the heart also expresses MTP, suggesting that the heart has the
capacity to secrete lipoproteins.
Human and mouse apoB gene expression was assessed with RNase
protection assays, as previously described.5 6 To
assess MTP expression, we probed Northern blots of human or mouse RNA
(Clontech) with a 32P- or digoxigenin-labeled
mouse MTP cDNA fragment.
Fixation, lipid staining, and sectioning of human heart tissue
for electron microscopy were performed as previously
described.7 In situ hybridization studies on
heart tissue from p158–human apoB transgenic mice were performed as
described previously.6
To determine which cells within the heart actually express the
human apoB gene, we performed in situ hybridization studies on the
hearts of two human apoB transgenic mice. A
35S-labeled antisense riboprobe yielded specific
staining of the myocardium in both the atrium and ventricle
(Figure 2
To explore the possibility that the heart might synthesize
lipoproteins, we examined human cardiac tissue by electron microscopy.
Interestingly, we observed lipid-staining small particles (
MTP has been shown to be essential for the secretion of apoB-containing
lipoproteins from cells. For example, HeLa cells, which lack MTP and
apoB gene expression, do not have the capacity to make
lipoproteins.9 When transfected with apoB cDNA
alone, HeLa cells synthesize apoB but cannot secrete lipoproteins. If
MTP is then expressed in the apoB-transfected HeLa cells, they acquire
the capacity to assemble apoB-containing lipoproteins and secrete them
into the medium. In the present study, we document that the heart
expresses both MTP and apoB, strongly suggesting that it has the
capacity to synthesize and secrete lipoproteins. To further
analyze that possibility, we performed ultrastructural studies
on human cardiac myocytes. These ultrastructural studies documented the
presence of small (
The finding that the apoB and MTP genes are expressed in two
species separated by more than 80 million years of mammalian evolution
suggests that the expression of these genes is important. However, the
reason why cardiac myocytes express the apoB and MTP genes has not yet
been defined. We suggest that the secretion of lipoproteins by the
heart may represent a pathway of "reverse
triglyceride transport," by which cardiac myocytes can
unload surplus fatty acids that are not required for fuel. The normal
heart uses large amounts of fatty acids for mitochondrial
ß-oxidation.10 Even though
triglyceride hydrolysis and the delivery of fatty acids to
the heart are regulated at multiple levels,10 it
is easy to imagine how rapidly changing metabolic demands
(eg, ischemia or even a sudden decrement in physical activity)
might cause heart myocytes to face an overly abundant supply of
intracellular fatty acids, which could be toxic to
myocytes.10 Although myocytes can store surplus
fatty acids in the form of cytosolic triglyceride droplets,
their capacity to store triglycerides is obviously not
unlimited.11 In the future, we believe that it
will be interesting to determine whether overexpression of apoB (ie, in
human apoB transgenic mice) might mitigate genetically or
environmentally induced fat accumulation within cardiac myocytes.
Homozygosity for null mutations in the MTP gene (as in
abetalipoproteinemia12) or apoB gene (as in
homozygous
hypobetalipoproteinemia13)
prevents the secretion of apoB-containing lipoproteins by the liver and
intestine and results in an accumulation of cytosolic fat droplets in
hepatocytes and absorptive enterocytes. Whether the absence
of cardiac lipoprotein secretion would result in an accumulation of
fats within the cardiac myocyte is not known. There have been reports
of cardiomyopathy and arrhythmias in
patients with abetalipoproteinemia,12 but no
comprehensive study of cardiac pathology in patients with either
syndrome has been reported. One approach for addressing the
consequences of absent lipoprotein secretion by the heart would be to
create heart-specific MTP or apoB knockout mice and then determine
whether the absence of lipoprotein secretion would lead to myocardial
fat accumulation.
Received December 2, 1997;
revision received February 23, 1998;
accepted February 25, 1998.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Genes for Apolipoprotein B and Microsomal Triglyceride Transfer Protein Are Expressed in the Heart
Evidence That the Heart Has the Capacity to Synthesize and Secrete Lipoproteins
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Expression
of both the apolipoprotein B (apoB) gene and the microsomal
triglyceride transfer protein (MTP) gene is required for
the assembly and secretion of triglyceride-rich
lipoproteins in the liver and intestine. Both genes have been assumed
to be silent in the heart. 
250 Å) within the Golgi
apparatus.
Key Words: apolipoproteins • lipoproteins • fatty acids • proteins • myocardium
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The B apolipoproteins
apoB48 and apoB100 play crucial structural roles in intracellular
assembly of the triglyceride-rich lipoproteins in both the
liver and the intestine.1 The assembly and
secretion of apoB-containing lipoproteins in liver and intestine is
absolutely dependent on microsomal triglyceride transfer
protein (MTP), which is thought to transfer lipids to apoB while the
apoB transcript is being translated, allowing apoB to fold correctly
and assemble a lipoprotein with a neutral lipid
core.2
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
RNA was prepared from mouse hearts and human heart tissue
with the Totally RNA kit (Ambion). Two samples of human heart ventricle
were obtained from the explanted hearts of cardiac transplant
recipients at California Pacific Medical Center, San Francisco,
according to a protocol approved by the Institutional Review Board. Two
additional samples of human heart tissue were provided by Drs K. Wyne
and H.H. Hobbs (University of Texas Southwestern Medical Center,
Dallas), and one sample of human heart RNA was purchased from
Clontech.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
We considered the possibility that the expression of human
apoB in the heart might represent an unusual artifact that
resulted from the expression of a fragment of human genomic DNA in the
mouse. We therefore assessed the expression of the
endogenous apoB gene in humans as well as in nontransgenic
mice. ApoB gene expression was easily detectable in each of the human
hearts (Figure 1A
). An RNase protection
assay revealed that the mouse apoB gene was also expressed in the
hearts of nontransgenic mice (Figure 1B). To determine whether the
heart expresses the MTP gene, we examined human and mouse heart RNA by
Northern blot analysis (Figure 1C). These studies revealed that
the MTP gene is expressed in the heart in both species, although at a
lower level than in the liver. Finding MTP gene expression in the heart
is consistent with an old observation by Wetterau and
Zilversmit: that microsomal vesicles isolated from hearts contained MTP
activity levels that were 3% of those in microsomal vesicles from the
liver.8
View larger version (35K):
[in a new window]
Figure 1. A, Phosphorimage showing RNase protection
assays with a 220-bp human apoB-specific 32P-riboprobe
demonstrating expression of apoB gene in human heart (60 µg RNA per
lane) and human liver (5 µg RNA). All lanes were exposed to the
phosphorimager screen for 16 hours. B, Autoradiogram
showing an RNase protection assay with a 245-bp mouse apoB-specific
32P-riboprobe demonstrating expression of mouse apoB in
heart (40 µg of RNA) and liver (5 µg of RNA) from nontransgenic
mouse. Lane showing liver expression was exposed for 8 hours; lane
showing heart expression was exposed for 48 hours. C, Northern blot
analysis showing MTP gene expression in human heart and liver
and mouse heart and liver. ).
View larger version (74K):
[in a new window]
Figure 2. In situ hybridization studies on heart
sections from human apoB transgenic mouse generated with a 145-kb BAC.
A and B, Dark-field microscopy of heart sections after in situ
hybridization with apoB antisense and sense 35S-riboprobes,
respectively.6 C, Hematoxylin-eosin–stained section of the
same heart. All sections are from the same area of heart, showing the
border between atrium and ventricle. The 35S-labeled apoB
antisense riboprobe yielded specific staining of myocardium
in both ventricle (thicker cardiac tissue) and atrium (thinner cardiac
tissue at top of each panel, above AV valve), with intensity of
staining being greater in atrium. Note absence of signal in AV valve
tissue in A. The bright circle in lower right corner of A is an
artifact of dark-field microscopy. 250 Å in
diameter) and particulate material within the Golgi
apparatus, suggesting that cardiac myocytes synthesize and
secrete lipoproteins (Figure 3).
View larger version (168K):
[in a new window]
Figure 3. Electron micrographs of cardiac myocyte
Golgi areas from two human hearts showing small lipid-staining
particles and particulate material within Golgi compartments. Smaller
solid arrows point to single discrete small lipid-staining particles
250 Å in diameter within Golgi-associated vesicles or tubules. Open
arrows point to lipid-staining particulate material within Golgi
stacks. Magnification x36 000.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In this study, we demonstrate that both the human heart and
the mouse heart express significant levels of apoB and MTP, two gene
products that are essential for the formation of
triglyceride-rich lipoproteins. In situ hybridization
studies indicated that human apoB mRNA is produced in the cardiac
myocytes. Reverse transcriptase–polymerase chain reaction
analysis of RNA from primary cultures of mouse cardiac myocytes
also has indicated that human apoB mRNA is produced in myocytes
(unpublished observations, T.V.-R. and I.J.G.). 250 Å in diameter) lipid-staining particles
within the Golgi apparatus of human heart myocytes, a
finding that is consistent with the presence of apoB-containing
lipoproteins in the secretory pathway.
Acknowledgments
This work was supported in part by NIH grants HL-41633 (to
Dr Young) and HL-45095 (to Dr Goldberg), a Grant-in-Aid from the
American Heart Association, Western States Affiliate (to Dr Hamilton
and J.S. Wong), a fellowship award from the American Heart Association,
Western States Affiliate (to Dr Nielsen), a grant from the Danish Heart
Association/Medical Research Council (to Dr Nielsen), and a fellowship
from the Deutsche Forschungsgemeinschaft (to Dr Raabe). We thank Drs K.
Wyne and H.H. Hobbs, University of Texas Southwestern Medical Center,
Dallas, and Dr D. Hill and N. Topic from California Pacific Medical
Center, San Francisco, for human heart tissues; L. Prentice for
preparing tissue sections for in situ hybridizations; and J. Wetterau
and D. Gordon for a mouse MTP cDNA clone.
Footnotes
Reprint requests to Stephen G. Young, MD, Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, PO Box 419100, San Francisco, CA 94141-9100.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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