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. 2013 Apr;54(4):1092-102.
doi: 10.1194/jlr.M034710. Epub 2013 Jan 23.

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Nina M Pollak  1 , Martina Schweiger, Doris Jaeger, Dagmar Kolb, Manju Kumari, Renate Schreiber, Stephanie Kolleritsch, Philipp Markolin, Gernot F Grabner, Christoph Heier, Kathrin A Zierler, Thomas Rülicke, Robert Zimmermann, Achim Lass, Rudolf Zechner, Guenter Haemmerle
Affiliations

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Nina M Pollak et al. J Lipid Res. 2013 Apr.

Abstract

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.

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Figures

Fig. 1.
Fig. 1.
CM-Plin5 provokes massive TG accumulation in CM of transgenic mice. A: A scheme depicting the Plin5 transgene used for microinjection and the generation of transgenic mice. The mouse Plin5 cDNA was cloned downstream of the murine CM-specific α-MHC promoter. For mRNA stabilization, 1.8 kb of the nontranslated human β-globin gene were inserted at the 3′ end of the Plin5 cDNA. The transgene fragment was flanked by NotI restriction enzyme sides. B: Cardiac Plin5 mRNA expression levels were highly increased in 12-week-old mice of two different transgenic lines (indicated as CM-Plin5 Tg26 and CM-Plin5 Tg32, respectively) compared with wt Plin5 mRNA expression levels (n = 5; ***P < 0.001). C: TG levels were drastically increased in CM of 12-week-old transgenic mice and the extent of TG accumulation correlated with Plin5 mRNA expression levels of the two transgenic lines (n ≥ 4; ***P < 0.001 compared with wt). D: Hearts from 12-week-old CM-Plin5 mice (from transgenic line 32 which was established for further characterization) were significantly heavier compared with wt hearts of nonfasted and fasted mice (n ≥ 4; ***P < 0.001). E: Cytosolic Plin5 protein levels were not significantly changed in CM of CM-Plin5 mice compared with wt. LD fraction could be merely prepared from CM of CM-Plin5 mice and contained large amounts of Plin5 protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control and cytosolic marker protein. F: Plin5 protein levels were comparable in mitochondria isolated from 12-week-old transgenic and wt mice, respectively. COXIV served as loading control for mitochondrial proteins. For Western blot analyses, equal protein amounts (10 μg) were separated by SDS-PAGE prior to blotting.
Fig. 2.
Fig. 2.
Measurement of tissue TG hydrolytic activities and FAs released from cardiac LDs. A: Cardiac TG hydrolase activities were increased in nonfasted and fasted 12-week-old Plin5 transgenic mice compared with the activity of wt tissues (n = 5; ***P < 0.001 versus wt; §§P < 0.01; §§§P < 0.001 versus nonfasted). B: Addition of recombinant GST-tagged CGI-58 significantly increased TG hydrolytic activities in both genotypes (n = 5; **P < 0.01; ***P < 0.001 versus wt; §P < 0.05; §§§P < 0.001 fasted versus nonfasted). C: Protein expression levels of ATGL and its coactivator CGI-58 were markedly elevated in CM of CM-Plin5 mice compared with wt (12-week-old mice). Analyzed samples contained 30 μg protein. Specific signals for ATGL and CGI-58 are indicated as arrows. GAPDH served as protein loading control. D: FA release of LD preparations isolated from CM of Plin5 transgenic and ATGL-deficient mice when incubated with COS-7 cell lysates containing LacZ, ATGL, ATGL + CGI-58, and HSL. The ATGL- and HSL-mediated FA release was significantly reduced in LD preparations of Plin5 transgenic mice compared with that of LDs from ATGL-deficient mice (n = 3). Data are shown as mean + SD. *P < 0.05; **P < 0.01; and ***P < 0.001 versus LDs incubated with LacZ containing lysates.
Fig. 3.
Fig. 3.
Ectopic Plin5 expression does not affect ATGL enzymatic activity but establishes a lipolytic barrier. A: TG hydrolase activity assays of mixtures of COS-7 cell lysates containing LacZ, ATGL, Plin5, and G0S2, respectively. TG hydrolytic activities were assayed with a micellar 3H-labeled triolein substrate. Data are shown as mean + SD of n = 3. ***P < 0.001 versus LacZ control. B: COS-7 cells transfected with lacZ, ATGL, and/or Plin5 were loaded with 400 μM oleic acid and 3H-labeled oleic acid as tracer and intracellular 3H-incorporation into the TG pool was determined. Data are shown as mean + SD of n = 3. *P < 0.05; **P < 0.01 versus LacZ controls. C: Western blot analyses of Plin5 protein levels in LDs prepared from Plin5-transfected COS-7 cells (LD-Plin5) and protein levels of LacZ, ATGL, CGI-58, G0S2, and HSL in cell lysates of COS-7 cells transfected with the respective cDNA expression plasmids. Analyzed cell lysates contained 10 μg protein. D: TG hydrolysis using isolated LDs as substrate. LDs were isolated from COS-7 cells transfected either with LacZ or Plin5 and loaded with oleic acid and 3H-labeled oleic acid as tracer. LD preparations were incubated with ATGL, ATGL + CGI-58, or HSL-containing COS-7 cell lysates and the release of 3H-FAs were measured. Data are mean + SD of n = 3. ***P < 0.001 versus LacZ-containing lysates and §§§P < 0.001 indicates significances among control versus Plin5-coated LDs. E: COS-7 cell lysates containing either LacZ, ATGL, or ATGL + CGI-58 were incubated with 3H-labeled triolein substrate. In some cases, LDs isolated from LacZ or Plin5-transfected cells were added to determine whether these LDs can compete with the TG substrate. Addition of LDs of LacZ, but not of Plin5 transfected cells largely inhibited hydrolysis of the 3H-triolein substrate suggesting that Plin5-coated LDs cannot compete with the micellar TG substrate. Data are shown as mean + SD of n = 3. **P < 0.01 and ***P < 0.001 versus LacZ samples. §P < 0.01 and §§§P < 0.001 indicate significances of control versus Plin5-enriched LDs.
Fig. 4.
Fig. 4.
Measurement of cardiac mRNA levels of established PPARα and PPARβ/δ target genes and mitochondrial CPT-1 activity. A: mRNA expression levels of PPARα, selected PPARα and PPARβ/δ target genes, PGC-1α and PGC-1β were determined by RT-qPCR in CM RNA prepared from 12-week-old fasted Plin5 transgenic and wt mice, respectively (n ≥ 5). *P < 0.05 and **P < 0.01 versus wt samples. B: Western blot and densitometric analysis of CPT-1 protein expression levels in mitochondria preparations of 15-week-old fasted mice. COXIV served as loading control (**P < 0.01). Ten micrograms of mitochondrial protein were separated by SDS-PAGE prior to blotting. C: CM-specific Plin5 overexpression significantly decreased CPT-1 activity in mitochondria preparations from CM of CM-Plin5 mice compared with that of controls (n ≥ 5; **P < 0.01 versus WT mice).
Fig. 5.
Fig. 5.
Transmission electron microscopy of CM sections revealed marked differences in cardiac morphology of Plin5 transgenic and ATGL-deficient mice. Cardiac tissue was prepared from mice after perfusion with paraformaldehyde. Fixed tissue sections were stained and examined by transmission electron microscopy. Cardiac sections from wt (A–C) showed a typical intermyofibrillar network and clusters of mitochondria. CM-Plin5 cardiac sections (D–F) showed hypertrophied LDs of similar size homogenously dispersed throughout the cytoplasm which seems not to interfere with the intermyofibrillar network. Most obviously, LDs were tightly associated with mitochondria. In contrast, cardiac sections obtained from ATGL-deficient mice [ATGL-knockout (ko) (G–I)] showed the accumulation of LDs of varying sizes including giant droplets. Overall, the cardiomyocyte architecture as well as the shape of mitochondria seemed to be markedly hampered by the giant droplets. Scale bars: 1 μm upper panel, 0.5 μm middle panel, 0.2 μm lower panel. m, mitochondria; mf, intermyofibrillar network.
Fig. 6.
Fig. 6.
mRNA expression levels of LPL, CD36, and PPARγ were reduced in CM of Plin5 transgenic mice whereas LPL activities were comparable to that of WT mice. A: mRNA expression levels were measured by RT-qPCR in cardiac tissue RNA derived from 12-week-old Plin5 transgenic and wt mice, respectively (n ≥ 4). **P < 0.01 and ***P < 0.001 versus WT mice. B: For the measurement of cardiac LPL-activities, hearts from overnight-fasted mice were surgically removed and minced in a medium containing heparin. LPL-activity of the supernatant was measured in duplicates. Values are shown as mean + SD of tissue samples from 4 mice of each genotype.
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