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
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.
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 (≈250 Å) within the Golgi apparatus.
Conclusions—These studies strongly suggest that the heart synthesizes and secretes apoB-containing lipoproteins.
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
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.
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.
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
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
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 (≈250 Å in diameter) and particulate material within the Golgi apparatus, suggesting that cardiac myocytes synthesize and secrete lipoproteins (Figure 3⇓).
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.).
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 (≈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.
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 β-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.
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.
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.
- Received December 2, 1997.
- Revision received February 23, 1998.
- Accepted February 25, 1998.
- Copyright © 1998 by American Heart Association
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