Fig 2: In vivo AAV9-mediated REEP5 depletion in mice results in increased cardiac fibrosis, activated cardiac ER stress, cardiac dysfunction, and death.a Immunoblot analysis of AAV9-mediated REEP5 depletion in mice 4 weeks post viral infection. b Histological analyses of AAV9 Scram shRNA and REEP5 shRNA injected mouse hearts at 4 weeks post viral infection. Top panels: H&E, bottom panels: Masson’s trichrome stain. Scale, 20 µm. All images shown are representative of approximately 20 total images captured per condition, n = 3 independent biological replicates. c Transmission electron microscopy analysis of AAV9 REEP5 shRNA-injected myocardium reveals degeneration of muscle fibers and disrupted SR membranes and organization compared to scram controls. M mitochondria, SR sarcoplasmic reticulum, V vacuoles. Scale, 0.5 nm. All images shown are representative of approximately 20 total images captured per condition, n = 3 independent biological replicates. d In vivo echocardiographic heart function assessment of AAV9 scram and AAV9 REEP5 shRNA-injected mice at 4 weeks post viral infection, n = 5. e Echocardiographic M-mode and B-mode measurements showed significantly compromised cardiac function with reduced cardiac ejection fraction and cardiac output, n = 5 independent biological replicates. Asterisks indicate a statistically significant p value in a Tukey’s multiple comparison analysis where ***p < 0.001; data are presented as mean ± SEM. f Immunoblotting analysis of cardiac ER stress markers (GRp78, GRp94, XBP1, ATF4, and CHOP) and ER-dependent apoptosis (caspase 12) upon AAV9-induced REEP5 depletion in vivo in mice, n = 3; p values are shown. g Immunoblots of RTN4, ATL3, and CKAP4 upon AAV9-induced REEP5 depletion in vivo in mice, n = 3 independent biological replicates. Source data containing original uncropped immunoblots are provided as a Source Data file.

Fig 3: Differential distribution of EGFP-tagged sheet/tubule proteins in N2a cells. (A) N2a cell line expressing EGFP-Climp63 (upper panels) or EGFP-DP1 (lower panels) (green) was stained with indicated marker proteins for ER and Golgi (red). Fluorescence intensities of the regions indicated by white lines are plotted on the bottom of the panels, and well co-distributions between green (EGFP) and red (markers) are indicated by arrowheads. Regions enriched for Climp63 and DP1/Rtn4 are also indicated in bottom lines with different colors. (B) Higher magnification of the cell body regions (boxes in the lower panels). Asterisks indicate the DP1/Rtn4-eiriched regions. Note the almost exclusive localizations of EGFP-Climp63 with ER tubule Rtn4, and EGFP-DP1 with sheet Climp63. Canx and KDEL were relatively well co-distributed with EGFP-Climp63 rather than EGFP-DP1, whereas GM130 was not co-distributed with them. Scale bars are 10 µm (A) and 4 µm (B).

Fig 4: Isolated murine aCMs can be genetically manipulated in vitro to functional effect.a Myocytes isolated from a PLN-null mouse were transfected using lentiviral vectors expressing wildtype (WT) and human pathogenic R9C phospholamban (PLN) flag tagged cDNAs. Immunoblots to anti-flag and a-tubulin. b Confocal images of aCMs stained for FLAG expression post lentiviral transfection for PLN. Scale bars represent 20 µm. c Traction force microscopy of aCMs post transfection. d Myocytes isolated from a WT mouse were transfected using AAV9 vectors encoding Reep5 shRNA sequence and assessed by immunoblots against REEP5 and tubulin. Reep5 KD treatment were loaded at higher volume to produce quantifiable bands. e Confocal images of aCMs confirm shRNA knockdown of Reep5 and resulting disorganization of SR (RyR2) and ER (KDEL motif) compared to scrambled shRNA control; scale bars represent 20 µm. f Peak Ca2+ transient amplitude is not significantly affected by Reep5 KD (N = 6). g Time to peak Ca2+ transient amplitude is significantly higher after Reep5 KD, but transient decay time to 50% is not affected. All data expressed as mean ± SEM; N denotes biological replicates; significance indicated by *(p < 0.05), **(p < 0.01), and ***(p < 0.001).

Fig 5: Proteomic analysis of the immunoprecipitates for mRFP-Climp63 and mRFP-DP1 isolated from N2a cells in a detergent-free condition. (A) Schematic procedure of immuno-isolation and proteomics of different ER domains. The cell lines were homogenized in detergent-free sucrose buffer and subjected to immunoprecipitation (IP), followed by Western blotting or LC-MS. (B) Western blotting of the immunoprecipitates by anti-mRFP antibodies (3G8 and 8D6) from N2a cell lines expressing mRFP, mRFP-Climp63 and mRFP -DP1. Precipitated proteins were analyzed by antibodies for indicated proteins (*non-specific bands). (C-E) Obtained immunoprecipitates were digested with trypsin and analyzed by LC-MS. Proteome Discoverer version 2.2 was used to identify the proteins and quantify their abundancies (label free quantification). After normalization with the amount of precipitated mRFP-tagged proteins, protein abundancies were compared between the two IP sets and log2 values of the fold changes (FCs; mRFP-Climp63-IP/mRFP-DP1-IP) were calculated. (C) Scatter plot of log2FCs for two IP sets (3G5 and 8D6). Proteins enriched by mRFP-Climp63-IP (red) or mRFP-DP1-IP (green) in two biological replicates (D) were colorized and known sheets and tubule proteins are indicated. (D) Identified proteins enriched in mRFP-Climp63-IP (upper diagram) or in mRFP-DP1-IP (lower diagram) in two biological replicates (rep 1 and 2); total 194 and 50 proteins were identified in both replicates, respectively. (E) Identified proteins were processed for functional annotation analysis. Proteins related to nucleus were relatively abundant in the mRFP-Climp63-IP, whereas proteins related to ER lumen, Golgi apparatus and chaperone were abundant in the mRFP-DP1-IP.