Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy

Abstract

Lipoprotein lipase is the principal enzyme that hydrolyzes circulating triglycerides and liberates free fatty acids that can be used as energy by cardiac muscle. Although lipoprotein lipase is expressed by and is found on the surface of cardiomyocytes, its transfer to the luminal surface of endothelial cells is thought to be required for lipoprotein lipase actions. To study whether nontransferable lipoprotein lipase has physiological actions, we placed an alpha-myosin heavy-chain promoter upstream of a human lipoprotein lipase minigene construct with a glycosylphosphatidylinositol anchoring sequence on the carboxyl terminal region. Hearts of transgenic mice expressed the altered lipoprotein lipase, and the protein localized to the surface of cardiomyocytes. Hearts, but not postheparin plasma, of these mice contained human lipoprotein lipase activity. More lipid accumulated in hearts expressing the transgene; the myocytes were enlarged and exhibited abnormal architecture. Hearts of transgenic mice were dilated, and left ventricular systolic function was impaired. Thus, lipoprotein lipase expressed on the surface of cardiomyocytes can increase lipid uptake and produce cardiomyopathy.

Figure 1

The hLpL^GPI DNA construction. (a) The human LpL minigene with a muscle creatine kinase promoter was used as a template DNA for generating the hLpL^GPI DNA construction. The translational start codon (ATG), stop codon (TGA), and the polyadenylation signal (PolyA) are indicated. MCK, muscle creatine kinase. (b) The hLpL^GPI DNA was inserted into the plasmid containing the CMV promoter for expression in transfected cells. (c) The hLpL^GPI DNA was subcloned into the plasmid containing the α-MHC promoter. The probe used for Northern blot analysis is indicated. kbps, kilobasepairs.

Figure 2

LpL activity in the medium. CHL cells were transfected with hLpL^GPI DNA. LpL activity in the medium released with heparin (10 U/ml) and/or PIPLC (2 U/ml) for 30 min at 37°C was measured. LpL activity was increased 4.6-fold with PIPLC and 8.1-fold with both PIPLC and heparin in the hLpL^GPI-CHL cells (black bars), compared with that in W-CHL cells (white bars). Values are expressed as means ± SD. *P < 0.01.

Figure 3

LpL expression in plasma and hearts. (a) Postheparin plasma LpL activity. There was no difference in postheparin LpL activity between LpL1 and three lines of hLpL^GPI/LpL1 mice. LpL1, n = 6; line 346, n = 3; line 357, n = 6; line 358, n = 2. (b) Heart LpL activity. Hearts from control and three lines of male transgenic animals were homogenized and assayed for LpL activity in triplicate. Homogenates of hearts of hLpL^GPI/LpL1 mice (line 357, n = 3) had 3.8-fold more LpL activity than control LpL1 mice (n = 4). *P < 0.01. (c) Myocardial human LpL. Human LpL was differentiated from mouse LpL using an mAb against human LpL activity. All the additional LpL activity in hearts from hLpL^GPI/LpL1 mice (line 357, n = 3) was inhibited by the Ab, and no inhibition was found when the Ab was added to homogenates from control LpL1 hearts (n = 4). The graph shows the amount of activity inhibited by the Ab. Values are expressed as means ± SD. *P < 0.01. (d) Northern blot analysis of hLpL^GPI mouse tissue RNA. Ten micrograms of total heart RNA from male mice was subjected to Northern blot analysis. Probe is shown in Figure 1c. The hLpL^GPI mRNA was detected only in the hearts. H, heart; M, skeletal muscle; A, adipose; Lu, lung; Li, liver; K, kidney; S, spleen. (e) Lipoprotein profiles of LpL1 and hLpL^GPI/LpL1 mice. Cholesterol distribution for LpL1 mice is shown with open circles and hLpL^GPI/LpL1 mice with filled circles.

Figure 4

VLDL turnover studies in hLpL^GPI/LpL1 mice. (a and b) Plasma VLDL clearance without (a) and after heparin injection (b) in LpL1 and hLpL^GPI/LpL1 mice. [³H]palmitate-labeled VLDL produced in LpL1 mice was intravenously injected into LpL1 and hLpL^GPI/LpL1 male mice, and plasma was obtained by retro-orbital bleeds. The plasma count at 0.5 min after injection was considered as the injected dose. Plasma VLDL clearance did not differ between LpL1 (open circles, n = 9) and hLpL^GPI/LpL1 mice (filled circles, n = 8). For FCR, LpL1 versus hLpL^GPI/LpL1 = 13.7 ± 6.6 versus 12.1 ± 4.4 pools/h. Heparinized hLpL^GPI/LpL1 mice (filled circles, n = 10) had faster clearance of radiolabeled VLDL than LpL1 mice (open circles, n = 9). For FCR, LpL1 versus hLpL^GPI/LpL1 15.3 ± 6.0 versus 24.3 ± 7.4 pools/h, P < 0.02. (c and d) Heart uptake of VLDL-TG (c) and after heparin (d) from LpL1 and hLpL^GPI/LpL1 mice. (c) Hearts of hLpL^GPI/LpL1 (black bar, n = 8) mice had 54% more VLDL-TG uptake than control (LpL1) hearts (white bar, n = 9). LpL1 versus hLpL^GPI/LpL1 = 0.016 ± 0.004 versus 0.025 ± 0.009 (heart dpm/injected dpm). (d) In the presence of heparin, hLpL^GPI/LpL1 mice (black bar, n = 10) had 26% more VLDL-TG uptake in the hearts compared with control LpL1 mice (white bar, n = 9). LpL1 versus hLpL^GPI/LpL1 = 0.015 ± 0.002 versus 0.019 ± 0.003 (heart dpm/injected dpm). Values are expressed as means ± SD. *P < 0.05; **P < 0.01.

Figure 5

Evidence for cardiomyopathy. (a) Heart size. Hearts were obtained from 4-month-old male mice, and the heart weight was divided by total body weight. The hLpL^GPI/LpL1 mice (filled circles, n = 11) had larger hearts (milligrams per gram body weight) than LpL1 mice (open circles, n = 11). *P < 0.02. (b and c) Survival. Breeding female hLpL^GPI/LpL1 mice (n = 11) died more frequently than LpL1 female mice (n = 14); males also died more rapidly. The hLpL^GPI/LpL1 males (n = 8) also died more rapidly than LpL1 male mice (n = 14). Differences among survival curves were compared using Kaplan-Meier survival analysis. *P < 0.01.

Figure 6

(a and b) LpL immunofluorescence in the hearts. An mAb was used to detect hLpL in hearts. In mice with transgenic expression of normal hLpL in the hearts on the homozygous LpL knockout background (He-LpL/LpL0) (28), hLpL was found in the cytoplasm and at the membrane. Hearts from hLpL^GPI/LpL1 mice had intense staining for LpL on the surface of cardiomyocytes. In a, intensity of red staining is amplified tenfold compared with b. (c and d) Myocardial lipid accumulation in 24 h–fasted hLpL^GPI/LpL1 mice. Oil red O staining shows an abundance of neutral lipid droplets within the cardiomyocytes of hLpL^GPI/LpL1 (d) mice compared with LpL1 (c) mice. ×400. (e and f) Electron microscopy. Ultrastructure of LpL1 mouse myocardial tissues exhibited normal morphological features with well-organized myofilaments and mitochondria (e). T tubules are not visible. The hLpL^GPI/LpL1 myocytes appeared severely distorted due to more mitochondria, irregular Z band of myofibrils, and dilated T tubules (f). (g) Northern blot analysis. Ten micrograms of total RNA were isolated from heart and subjected to Northern blot analysis using a part of cDNA encoding PPARα, CPT-1, ACO, ANF, and GLUT4 as probes. GAPDH is shown as a control for loading.

Figure 7

Two-dimensional echocardiogram. Representative echocardiographic images from LpL1 (a and b) and hLpL^GPI/LpL1 (c and d) mice at end-diastole (left; a and c) and end-systole (right; b and d). The left ventricles of hLpL^GPI/LpL1 6-month-old female mice were significantly larger than control LpL1 mice (see Table 1).

Figure 8

Mechanisms of LpL mediated lipid uptake from TG-rich particles in the heart. (a) Cardiomyocytes express LpL that dissociates from the cell surface and migrates to the luminal surface of capillary endothelial cells. At this location, LpL attaches to heparan sulfate proteoglycans (HSPG) and is able to interact with circulating TGRP. FFAs are released that cross the endothelial barrier and are acquired by myocytes. (b) Our studies show that LpL associated with the cardiomyocyte surface, in this case via a GPI anchor, also promotes lipid uptake. For this to occur, some TGRP, perhaps lipoproteins that are partially digested by endothelial-associated LpL, must exit the vasculature, enter the subendothelial space, and directly interact with cardiomyocytes. It should be noted that LpL is found on both endothelial and cardiomyocyte surfaces. Thus, the LpL-mediated interactions shown in both a and b are likely to occur in the hLpL^GPI mice and may play a role in normal physiology.