Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts

Abstract

Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling and facilitates free fatty acid (FA) uptake by interacting with several FA transport proteins. We previously reported that Cav3 knockout mice (Cav3KO) develop cardiac hypertrophy with diminished contractile function; however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that the uptake and oxidation of FAs and glucose were normal in hypertrophic Cav3KO hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including FA translocase (CD36) and carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly, myocardial cAMP content was significantly increased by 42%; however, this had no effect on PKA activity in Cav3KO hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a fourfold increase in the expression of cellular retinol binding protein-III and a 3.5-fold increase in 17beta-hydroxysteroid dehydrogenase type 11, a member of the short-chain dehydrogenase/reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, a loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, a loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. Thus we have identified a number of new target genes associated with the pathogenesis of cardiac hypertrophy.

Fig. 1.

Cardiac uptake of ¹⁴C-palmitate-BSA (A), ³H-triolein from Intralipid emulsion (B), and 2-deoxy-d-[1-¹⁴C]glucose (C) in hearts from 5- to 6-mo-old caveolin-3 knockout (Cav3KO; white bars; n = 10) was comparable with wild-type (black bars; n = 10) male mice. Data are expressed as a percentage of wild-type ±SE.

Fig. 2.

Hearts from 5- to 6-mo-old wild-type (black bars; n = 5) and Cav3KO (white bars; n = 5) male mice were homogenized and assayed for lipoprotein lipase (LPL) activity (A) and LPL mass (B). Values are expressed as means ± SE.

Fig. 3.

Myocardial substrate utilization was measured in 5- to 6-mo-old male wild-type (black bars; n = 5) and Cav3KO (white bars; n = 5) mice. Loss of Cav3 did not significantly alter glycolysis (A), glucose oxidation (B), and myocardial palmitate oxidation rates (C). Values are expressed as means ± SE.

Fig. 4.

Quantitative RT-PCR analysis of genes involved in fatty acid metabolism and glucose uptake. The expression of fatty acid translocase (CD36), fatty acid transport protein (FATP)1, FATP6, acyl-CoA oxidase (ACO), carnitine palmitoyltransferase 1 (CPT-1), peroxisome proliferator-activated receptor (PPAR)α, PPARδ, glucose transport protein (GLUT)1, and GLUT4 in hearts from 5- to 6-mo-old male Cav3KO mice (white bars; n = 9) were comparable with wild-type mice (black bars; n = 8). Gene expression was normalized to cyclophilin. Values are expressed as means ± SE. *P < 0.05. dwt, dry weight.

Fig. 5.

cAMP levels (A) were significantly elevated in hearts from Cav3KO (white bars; n = 5) compared with wild-type (black bars; n = 6) mice. However, PKA activity (B) in Cav3KO hearts (n = 5) was comparable with wild-type (n = 5) hearts. Values are expressed as means ± SE. *P < 0.05.

Fig. 6.

Loss of Cav3 (white bars; n = 7) increased cardiac triglyceride (A) and free FA (FFA; B) levels while decreasing total cholesterol (C) content (wild-type; black bars; n = 6). Glycogen levels (D) in hearts from Cav3KO mice (n = 7) were comparable with those of wild-type hearts (n = 7). Values are expressed as means ± SE. *P < 0.05; **P < 0.01.

Fig. 7.

Microarray data were confirmed by real-time PCR. Hearts from 5- to 6-mo-old Cav3KO male mice (white bars; n = 5) had significantly increased gene expression for cellular retinol binding protein (Rbp)7, 17β-hydroxysteroid dehydrogenase type 11 (HSD17β11), and Rab4a vs. wild-type (black bars; n = 5). The expression of aldolase B was markedly reduced in Cav3KO hearts. Values are expressed as means ± SE. *P < 0.05.

Fig. 8.

Immunohistochemical analysis revealed infiltration of CD45⁺ cells in fibrotic lesions in Cav3KO hearts. Paraffin-embedded Cav3KO (A and C) and wild-type (B and D) mouse hearts from 5- to 6-mo-old mice were subjected to immunostaining. Interestingly, CD45, a leukocyte marker, was only observed in perivascular fibrotic lesions in Cav3KO hearts (arrows). Interstitial and perivascular fibrosis and leukocyte staining were not observed in wild-type hearts (B and D).