Caveolin-1/3 double-knockout mice are viable, but lack both muscle and non-muscle caveolae, and develop a severe cardiomyopathic phenotype

Abstract

The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.

Figure 1.

Caveolin-1/3 dKO mice are deficient in the expression of all three members of the caveolin gene family (Cav-1, -2, and -3). Protein expression of caveolin family members was examined in wild-type and Cav-1/3 dKO mice. Note that in wild-type mice, fat and lung tissues abundantly express Cav-1 and Cav-2 proteins, while Cav-3 is abundantly expressed in striated muscle tissues, and all three caveolin family members are expressed in the vasculature (as in the aorta). Immunoblot analysis using isoform-specific caveolin mAb probes demonstrates a loss of Cav-1 and Cav-3 protein expression in all of the Cav-1/3 dKO tissues examined. Note that as Cav-2 requires Cav-1 for stabilization, oligomerization, and plasma membrane localization, Cav-2 protein expression is also dramatically down-regulated (by ∼95%) in Cav-1/3 dKO mice.

Figure 2.

Cav-1/3 dKO mice exhibit a loss of morphologically identifiable caveolae in both muscle and non-muscle cell types. To assess the consequence of a loss of Cav-1, -2, and -3 protein expression, muscle and non-muscle cells that normally show abundant caveolae were examined by transmission electron microscopy. Note that wild-type lung endothelia (A), adipocytes (B), skeletal muscle fibers (C), and aortic smooth muscle cells (D) all contain numerous caveolae, located at or near the plasma membrane (arrows). In contrast, both muscle and non-muscle cells derived from Cav-1/3 dKO mice show a loss of caveolae. RBC, red blood cell; LD, lipid droplet; ECM, extracellular matrix; PM, plasma membrane; Cyto, cytoplasm. Scale bar, 500 nm.

Figure 3.

Dual ablation of both Cav-1 and Cav-3 genes is required to disrupt caveolae formation in both cardiac myocytes and their adjacent cardiac endothelial cells. Tissue-specific formation of caveolae is dictated by the caveolin isoform expressed. Arrowheads point to Cav-1-induced caveolae, while arrows point to caveolae induced by Cav-3 expression. Note that a Cav-1 deficiency (Cav-1 KO) leads to a selective loss of caveolae in cardiac endothelial cells, but does not affect caveolae formation in cardiac myocytes (B). Conversely, a Cav-3 deficiency (Cav-3 KO) leads to a loss of caveolae in cardiac myocytes, while caveolae in the cardiac endothelial cells remain unaffected (C). Therefore, a loss of both Cav-1 and Cav-3 expression is necessary to completely abolish caveolae formation in cardiac tissue (D). Endo, endothelial cell. Scale bar, 500 nm.

Figure 4.

Cav-1/3 dKO mice exhibit cardiac hypertrophy as assessed by gated MRI. Representative short axis (transverse) images at the mid-level of the heart of WT (A) and Cav-1/3 dKO (B) during diastole. Note the concentric hypertrophy of the left ventricle (LV) in the Cav-1/3 dKO heart. RV, right ventricle; LV, left ventricle.

Figure 5.

Cav-1/3 dKO hearts histologically exhibit cardiac myocyte hypertrophy and disorganization, with features of chronic inflammation and fibrosis. Representative H&E stained sections of the myocardium of wild-type mice (WT) and Cav-1/3 dKO mice (dKO) are shown at various magnifications. Micrographs were taken with different objectives (10×, 20×, 40×, or 60×), as indicated. A, C, E, and G represent wild-type cardiac histology. B, D, F, and H show Cav-1/3 dKO cardiac histology, with marked cardiac myocyte disorganization. In B and D there is chronic inflammation marked by increased cellular infiltrates and fibrosis (arrows). F demonstrates an area of marked cardiac myocyte hypertrophy and disarray. H shows cardiac myocyte hypertrophy, interspaced with areas of cardiac myocyte degeneration, ie, myocytolysis (arrows).

Figure 6.

Caveolin-1/3 dKO ventricular tissue exhibits a switch to fetal programming, as demonstrated by an up-regulation of atrial natriuretic peptide (ANF). Cav-1/3 dKO and WT ventricular tissue was carefully separated from atrial tissue by dissection, and total RNA was extracted for Northern blot analysis. Note that the ANF transcript is highly up-regulated in the Cav-1/3 dKO ventricles, but absent in wild-type control ventricular tissue. Thus, these data provide further molecular evidence that Cav-1/3 dKO mice exhibit a hypertrophic cardiomyopathic phenotype.