Subcellular proteomic analyses reveal REEP5 knockdown in the mouse heart disrupts mitochondrial networks

Fig. 1. Overview of REEP5 in vivo cardiac knockdown and expression analysis. A, schematic of rAAV9-mediated delivery of REEP5-targeting shRNA or scrambled control via intraperitoneal injection at P7. Knockdown efficiency was assessed 1-4-weeks post-injection. Image created with BioRender.com. B, Top: immunoblot of REEP5 in cardiac tissues 1-4-weeks post-rAAV9 injection. Bottom left: densitometric quantification of REEP5 expression normalized to Actin loading control (n = 3–8 biological replicates; ∗∗∗p < 0.001 by one-way ANOVA with Tukey’s post-hoc test). Bottom right: qPCR analysis of Reep1–6 mRNA expression in Scramble and Reep5-KD tissues, normalized to Tbp (n = 4 biological replicates per group; ∗∗∗∗p < 0.0001 by one-way ANOVA with Tukey’s post-hoc test). C, immunofluorescence of REEP5 (green), RYR2 (purple) and Hoechst (blue) in cardiac tissues 4-weeks post-rAAV9 injection (the scale bar represents 20 μm). D, immunofluorescence of REEP5 (green), RYR2 (purple) and Hoechst (blue) in aCMs 4-weeks post-rAAV9 injection (the scale bar represents 10 μm). E and F, magnified view and profile plots of REEP5 and α-actinin across an 8 μm line scan; yellow line indicates scan region (the scale…

Fig. 2. Cardiac subcellular proteomics workflow and dataset validation. A, overview of organelle enrichment and proteomics workflow for cardiac tissues harvested 4-weeks post-rAAV9 injection. Image created with BioRender.com. B, venn diagram depicting identified proteins from the 3 organelle groups and pairwise comparisons for each. C, immunoblot validation of Calnexin , COX20 and GAPDH expression in microsomal, mitochondrial and cytosolic fractions. D, sankey plot showing the association of proteins identified in each subcellular fraction with major cardiac cell types based on integration with single-cell RNA-sequencing data from the adult mouse heart (19).

Fig. 3. Changes in the microsomal proteomic profile following Reep5 knockdown. A, principal component analysis and (B) volcano plot of the microsomal-enriched fraction. The volcano plot displays –log10p-values (two-tailed Student’s t- test) versus the difference in mean log2-transformed normalized protein intensities, highlighting proteins altered in the microsomal compartment following Reep5 knockdown. Highlighted in blue (downregulated) and orange (upregulated) are proteins with significant changes (FDR <0.05) in their expression following Reep5 knockdown. REEP5 is highlighted in black. C, Euclidean distance-based hierarchical clustering of significantly altered proteins (two-tailed Student's t-test, FDR < 0.05) resulting from Reep5 knockdown. Data represents mean average of Z-score normalized values. D, targeted heatmap of selected significantly altered microsomal proteins (two-tailed Student's t-test, FDR < 0.05). Data represents mean average of Z-score normalized values. E and G, Dot plots showing the top 12 enriched up and downregulated GO terms for biological process (E), cellular component (F) and molecular function (G). Dot size represents the number of genes associated with…

Fig. 4. Changes in the mitochondrial proteomic profile following Reep5 knockdown. A, principal component analysis and (B) volcano plot of the mitochondrial-enriched fraction. The volcano plot shows –log10p-values (two-tailed Student’s t-test) plotted against the difference in mean log2-transformed normalized protein intensities, reflecting Reep5-dependent changes in mitochondrial protein expression. Highlighted in blue (downregulated) and orange (upregulated) are proteins with significant changes (FDR < 0.05) in their expression following Reep5 knockdown. C, Euclidean distance-based hierarchical clustering of significantly altered proteins (two-tailed Student's t-test, FDR < 0.05) resulting from Reep5 knockdown. Data represents mean average of Z-score normalized values. (D) Targeted heatmap of selected significantly altered mitochondrial proteins (two-tailed Student's t-test, FDR < 0.05). Data represents mean average of Z-score normalized values. E and G, Dot plots showing the top 12 enriched up and downregulated GO terms for biological process (E), cellular component (F) and molecular function (G). Dot size represents the number of genes associated with each term, and color intensity…

Fig. 5. Changes in the cytosolic proteomic profile following Reep5 knockdown. A, principal component analysis and (B) volcano plot of the cytosolic-enriched fraction. The volcano plot presents –log10p-values (two-tailed Student’s t-test) versus the difference in mean log2-transformed normalized protein intensities, revealing cytosolic expression shifts following Reep5 knockdown. Highlighted in blue (downregulated) and orange (upregulated) are proteins with significant changes in their expression (FDR < 0.05) following REEP5 knockdown. C, Euclidean distance-based hierarchical clustering of significantly altered proteins (two-tailed Student's t -est, FDR < 0.05) resulting from Reep5 knockdown. Data represents mean average of Z-score normalized values. D, targeted heatmap of selected significantly altered cytosolic proteins (two-tailed Student's t-test, FDR < 0.05). Data represents mean average of Z-score normalized values. E and G, Dot plots showing the top 12 enriched up and downregulated GO terms for biological process (E), cellular component (F) and molecular function (G). Dot size represents the number of genes associated with each term, and colour intensity corresponds to the q-value.…

Fig. 6. Cardiomyocyte analysis of mitochondrial morphology and function following Reep5 knockdown. A, left: confocal imaging of mitochondrial structure in isolated aCMs stained with MitoTracker (the scale bar represents 10 μm). Right: zoomed-in view of boxed region from the left panel; arrows highlight individual mitochondria (the scale bar represents 5 μm). B and D, quantitative analysis of mitochondrial morphology based on MitoTracker imaging: (B) mitochondrial size (μm3), (C) number of mitochondria per cell normalized to cell volume, and (D) total cell volume (μm3) (n = 32–49 cells across 3 mice per group; ∗∗∗p = 0.0001, ∗∗∗∗p < 0.0001, unpaired Student’s t-test with Welch’s correction). E, confocal imaging of ROS levels in aCMs using CellROX (the scale bar represents 10 μm). F, quantification of CellROX fluorescence intensity normalized to cell area (n = 40 cells across 3 mice per group; ∗p = 0.0187, unpaired Student’s t-test with Welch’s correction).

Supplemental Fig. S1. 3D reconstruction of aCMs stained with REEP5 (green) and α-actinin (purple), imaged by confocal microscopy (scale bar, 7 µm).

Supplemental Fig. S2. (A–C) Immunoblot and quantification of aCMs probed for REEP5, MFN2 and GAPDH (n = 4 biological replicates, ∗ p < 0.05, unpaired Student’s t-test with Welch’s correction). (D) Immunofluorescence of TOMM20 (green) and SERCA2a (purple) imaged by confocal microscopy (scale bar, 10 µm).

Supplemental Fig. S3. 3D reconstruction of Scramble aCMs stained with MitoTracker, imaged by confocal microscopy (scale bar, 10 µm). Full 3D Movie available.

Supplemental Fig. S4. 3D reconstruction of Reep5-KD aCMs stained with MitoTracker, imaged by confocal microscopy (scale bar, 10 µm). Full 3D Movie available.