Popeye domain containing 1 (Popdc1/Bves) is a caveolae-associated protein involved in ischemia tolerance

Abstract

Popeye domain containing1 (Popdc1), also named Bves, is an evolutionary conserved membrane protein. Despite its high expression level in the heart little is known about its membrane localization and cardiac functions. The study examined the hypothesis that Popdc1 might be associated with the caveolae and play a role in myocardial ischemia tolerance. To address these issues, we analyzed hearts and cardiomyocytes of wild type and Popdc1-null mice. Immunoconfocal microscopy revealed co-localization of Popdc1 with caveolin3 in the sarcolemma, intercalated discs and T-tubules and with costameric vinculin. Popdc1 was co-immunoprecipitated with caveolin3 from cardiomyocytes and from transfected COS7 cells and was co-sedimented with caveolin3 in equilibrium density gradients. Caveolae disruption by methyl-β-cyclodextrin or by ischemia/reperfusion (I/R) abolished the cellular co-localization of Popdc1 with caveolin3 and modified their density co-sedimentation. The caveolin3-rich fractions of Popdc1-null hearts redistributed to fractions of lower buoyant density. Electron microscopy showed a statistically significant 70% reduction in caveolae number and a 12% increase in the average diameter of the remaining caveolae in the mutant hearts. In accordance with these changes, Popdc1-null cardiomyocytes displayed impaired [Ca(+2)]i transients, increased vulnerability to oxidative stress and no pharmacologic preconditioning. In addition, induction of I/R injury to Langendorff-perfused hearts indicated a significantly lower functional recovery in the mutant compared with wild type hearts while their infarct size was larger. No improvement in functional recovery was observed in Popdc1-null hearts following ischemic preconditioning. The results indicate that Popdc1 is a caveolae-associated protein important for the preservation of caveolae structural and functional integrity and for heart protection.

Figure 1. Localization of Popdc1 in WT cardiomyocytes.

(a) Popdc1 (red labeling) is evident at the lateral membrane (arrow), intercalated discs (arrowhead) and transverse striations (double arrow). (b) Upper panel, co-labeling for Popdc1 (red), Cav3 (green) and vinculin (light blue); on the left, merge of Popdc1 with Cav3 (white) and on the right, merge of Popdc1 with vinculin (white). Lower panel, 3D surface analysis based on Z-stack pictures. Popdc1 (red), Cav3 (light blue), vinculin (green), merge of Popdc1 with Cav3 (violet), merge of Popdc1 with vinculin, yellow. Square size 5×5 µm. (c) Popdc1 (red) and Cav3 (green) co-localize (yellow) in the sarcolemma (arrowhead) and T-tubules (arrows) in isolated adult WT cardiomyocytes (Adult CM), at sites of cell-cell contacts in cultured neonatal WT cardiomyocytes (CMC), and in Popdc1/Cav3 co-transfected COS7 cells at sites of cell adhesion (Asterisk) and inside the cells.

Figure 2. Co-immunoprecipitation of Popdc1 and Cav3.

(a) Immunoblots demonstrating the precipitation of Popdc1 and Cav3 using mouse anti-Cav3 antibodies (left) and rabbit anti Popdc1 antibodies (right). Top labels: IP: immunoprecipitation tool; Cav3, anti-Cav3; mIgG, mouse immunoglobulins (control); Popdc1, anti-Popdc1 antibodies; rIgG, rabbit immunoglobulins (control). Left/right labels: Popdc1 migration at 68 kDa and Cav3 migration at 22 kDa. (b) Co-immunoprecipitation of Popdc1 and Cav3 from COS7 cells co-transfected with Cav3 and full length (WT) or deletion mutants (Δ92 and Δ116) Myc-tagged Popdc1 expression constructs. The scheme depicts the position of the deletions relative to the predicted Cav3 binding site (BS). Shown is a Western immunoblot probed for the detection of Cav3. Input, Cav3 in the original cell extract; IP-Myc, Cav3 that was precipitated by the anti-Myc antibodies. The figure depicts results of a representative membrane.

Figure 3. Popdc1 density sedimentation in discontinuous sucrose gradients.

(a) Western blot analysis of gradient fractions. Fraction 1 and fraction 12 represent the lowest and highest density, respectively. [+/+], WT; [−/−], Popdc1-null; MβCD, methyl-β-cyclodextrin; I/R, ischemia/reperfusion; Cx43, connexin43. The molecular weights of the reactive bands were ∼68 kDa, ∼22 kDa, and ∼43 kDa, for Popdc1, Cav3, and Cx43, respectively. (b) The relative distribution of cholesterol and Cav3 throughout gradient fractions 1–9. *P<0.05, Cav3 in fraction 5, Popdc1-null vs. WT; ^#P<0.05, cholesterol in fraction 5, Popdc1-null vs. WT.

Figure 4. Popdc1 and Cav3 distribution in normal, mutant and injured hearts.

Confocal microscopy images depicting Popdc1 (red) and Cav3 (green); co-localization sites appear in yellow. Note the low staining intensity of Cav3 cross striations in the Popdc1-null and MβCD-treated hearts, and the disappearance of Popdc1 and Cav3 co-localization following I/R.

Figure 5. LTCC density sedimentation and [Ca²⁺]_i transients in Popdc1-null cardiomyocytes.

(a) Representative WB depicting the sedimentation pattern of LTCC (Cav1.2a, ∼200 kDa) and Cav3 (∼22 kDa) in membrane preparations of WT [+/+] and Popdc1-null [−/−] hearts. (b) Quantification of the relative distribution of LTCC and Cav3 in gradient fractions 5 (Fr5) and 6 (Fr 6) of the two genotypes. The results summarize, in arbitrary units (a.u.), three and five independent experiments for LTCC and Cav3, respectively. *P<0.05, fraction 5 vs. fraction 6 within each genotype. (c) Left, [Ca²⁺]_i transients in representative cardiomyocytes; Right, summary of the measurements performed; n = number of cells; Mean ± SEM.

Figure 6. Caveolae are altered in Popdc1-null hearts.

Electron micrographs showing caveolae (arrows) in WT and Popdc1-null hearts. Scale bar, 0.5 μm. In the bottom panel, summary of caveolae abundance and size measured along 680 and 660 mm membrane length in WT and Popdc1-null hearts, respectively; n = 3/genotype; Mean ± SEM; *P<0.00001.

Figure 7. Heart function and infarct size.

Hearts were subjected to Langendorff-perfusion as detailed in the Methods. (a) The LVP recovery during reperfusion starting at 1 min reperfusion. (b) Summary of LV function parameters at 30 min reperfusion. (c) Infarct size expressed as percent of the area at risk. *P<0.05, Popdc1 ^[−/−] vs. Popdc1 ^[+/+]; ^#P<0.05, Popdc1 ^[−/−] vs. Popdc1 ^[+/−]. In the curve comparison (ANOVA with multiple repeats), ^¶P<0.05, Popdc1 ^[+/+] vs. Popdc1 ^[−/−] and ^§P<0.05. Popdc1 ^[+/−] vs. Popdc1 ^[−/−]. Mean ± SEM; in (a) and (b) N = 12/group; in (c), N = 5/group.

Figure 8. Popdc1 is down regulated by I/R.

(a) and (b) RT-qPCR of Popdc1-3 and LacZ mRNAs from hearts isolated upon heart removal (Basal), at the end of stabilization (Stab) and at 90 min reperfusion (I/R). N = 5/group. AU, arbitrary units, Mean ± SEM; *P<0.05 compared to Basal. (c) Western blots of Popdc1 in WT hearts (Popdc1, ∼68 kDa; Actin, ∼42 kDa). (d) Cytochemical staining of LacZ activity (blue nuclei). Note, a higher intensity in subendocardial cells (white asterisk). Eosin counterstaining illustrates the general morphology. Images were captured at X50 and X100 magnification, as specified.

Figure 9. No preconditioning in Popdc1-null hearts and cardiomyocytes.

(a) WT and mutant hearts underwent I/R perfusion with and without IPC. Parameters of LV function registered at 30 min reperfusion are shown. Labels are as in Figure 1. Mean ± SEM; *P<0.05, I/R vs. IPC-I/R within a genotype. (N = 5–9/group). (b) Wild type and mutant cardiomyocytes were preconditioned with isoflurane (1.5%) or left untreated, then loaded with calcein-AM and exposed to H₂O₂ (200 µM). The change in calcein fluorescence with time of H₂O₂ treatment is shown. Summary of three experiments performed in triplicates. *P<0.05, WT (Popdc1 ^[+/+]) cells, isoflurane vs. untreated control. ^#P<0.05, control Popdc1 ^[−/−] vs. control Popdc1 ^[+/+] (isoflurane-untreated cells, open symbols). In the curve comparisons (ANOVA with multiple repeats), ^§P<0.05, Popdc1 ^[+/+] isoflurane vs. Popdc ^[+/+] control; ^¥P<0.05 Popdc1 ^[+/+] control vs. Popdc1 ^[−/−] control.