Loss of PDZK1 causes coronary artery occlusion and myocardial infarction in Paigen diet-fed apolipoprotein E deficient mice

Abstract

Background: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression ( approximately 95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol ('Western') diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI.

Principal findings: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic 'Paigen' diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33% increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36% increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375% increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle.

Conclusions: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful for studying coronary heart disease and suggest that PDZK1 may represent a valuable target for therapeutic intervention.

Figure 1. Immunoblot analysis of hepatic SR-BI expression.

Mice with the indicated genotypes were fed a high fat/high cholesterol/cholate-containing “Paigen” diet for three months. Livers were harvested and subjected to immunoblotting using anti-SR-BI and anti-actin (loading control) antibodies as described in Materials and Methods.

Figure 2. Lipoprotein cholesterol profiles from apoE KO and PDZK1/apoE dKO mice.

Plasma harvested from individual mice fed a Paigen diet for three months was size fractionated using FPLC, and the total cholesterol contents of the fractions (mg/dL plasma) were determined by enzymatic assay. Profiles averaged from 2 independent experiments for each genotype, each composed of pooled plasma from six apoE KO (open circles) and six PDZK1/apoE dKO (filled circles) mice per experiment are shown. Approximate elution positions of human VLDL, IDL/LDL and HDL are indicated.

Figure 3. Aortic root atherosclerosis in Paigen diet-fed apoE KO and PDZK1/apoE dKO mice.

Hearts were harvested from Paigen diet-fed apoE KO (A, C–D) and PDZK1/apoE dKO (B, E–F) mice as described in Methods (n = 8 per genotype). Left top panels: A–B: representative cross-sections of Oil red O-stained aortic root lesions. (magnification, ×20). Right top panel: quantification of aortic root atherosclerosis by planimetry. Unpaired Student's t-test was used to determine statistical significance. Bottom panels: immunohistochemistry of aortic root atherosclerotic plaques using CD68 (C and E) or alpha-smooth muscle actin α-SMA) (D and F) antibodies show that macrophages compose the overwhelming cell population of aortic root atherosclerotic plaques and that smooth muscle cells are rare in both apoE KO (C–D) and PDZK1/apoE dKO (E–F) mice. “L” indicates the vascular lumen, arrows indicate representative positive cells (magnification, ×100).

Figure 4. Effects of loss of PDZK1 on coronary atherosclerosis and cardiac fibrosis in apoE KO mice.

Hearts were harvested from Paigen diet-fed mice as described in Methods (n = 8 per genotype). Left panels: A–B: representative cross-sections of Oil red O-stained (A–B) or trichrome-stained (C) myocardial coronary arterioles, showing unremarkable arterioles in apoE KO (A) and totally occluded arterioles in PDZK1/apoE dKO (B–C) mice (magnification, ×100). The Oil red O stain shows that the coronary arteriole is occluded almost exclusively by lipid-rich lesions (B), while the trichrome stain shows that the arteriole is surrounded by fibrosis in an area of myocardial infarction in a PDZK1/apoE dKO mouse (C). D–E: trichrome stained sections of hearts showing areas of infarction/fibrosis stained blue in PDZK1/apoE dKO (E), while they are absent in apoE KO (D) mice (magnification, ×10). Right top panel: quantification of coronary artery occlusions in apoE KO and PDZK1/apoE dKO mice. Statistically significant differences by ANOVA Tukey posthoc test comparing the two genotypes within a given group are indicated as: P<0.001. Right bottom panel: quantification of cardiac fibrosis in apoE KO and PDZK1/apoE dKO mice. Unpaired Student's t-test was used to determine statistical significance.