Localization of Popdc1 in WT cardiomyocytes.

<p>(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.</p

Co-immunoprecipitation of Popdc1 and Cav3.

<p>(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.</p

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

<p>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 <i>Popdc1</i>-null and MβCD-treated hearts, and the disappearance of Popdc1 and Cav3 co-localization following I/R.</p

Popdc1 is down regulated by I/R.

<p>(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.</p

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

<p>(a) Representative WB depicting the sedimentation pattern of LTCC (Cav1.2a, ∼200 kDa) and Cav3 (∼22 kDa) in membrane preparations of WT [+/+] and <i>Popdc1</i>-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.</p

No preconditioning in <i>Popdc1</i>-null hearts and cardiomyocytes.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071100#pone-0071100-g001" target="_blank">Figure 1</a>. 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 (<i>Popdc1 </i>^[+/+]) cells, isoflurane vs. untreated control. ^#P<0.05, control <i>Popdc1 </i>^[−/−] vs. control <i>Popdc1 </i>^[+/+] (isoflurane-untreated cells, open symbols). In the curve comparisons (ANOVA with multiple repeats), ^§P<0.05, <i>Popdc1 </i>^[+/+] isoflurane vs. <i>Popdc </i>^[+/+] control; ^¥P<0.05 <i>Popdc1 </i>^[+/+] control vs. <i>Popdc1 </i>^[−/−] control.</p

Caveolae are altered in <i>Popdc1</i>-null hearts.

<p>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.</p

Heart function and infarct size.

<p>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, <i>Popdc1</i>^[−/−] vs. <i>Popdc1</i>^[+/+];^#P<0.05, <i>Popdc1</i>^[−/−] vs. <i>Popdc1</i>^[+/−]. In the curve comparison (ANOVA with multiple repeats), ^¶P<0.05, <i>Popdc1</i>^[+/+] vs. <i>Popdc1 </i>^[−/−] and ^§P<0.05. <i>Popdc1 </i>^[+/−] vs. <i>Popdc1</i>^[−/−]. Mean ± SEM; in (a) and (b) N = 12/group; in (c), N = 5/group.</p

Basal heart performance.

<p>Measurements taken at 30 min of normoxic Langendorff-perfusion (stabilization). LVP, left ventricular developed pressure; +dP/dt, rate of pressure development; −dP/dt, rate of pressure relaxation. HR, heart rate; RPP, rate⋅pressure product; CF, Coronary flow; Brackets, number of animals. Mean ± SEM.</p