DOI: 10.5061/dryad.1c59zw462
title: Data from: The deubiquitinase USP5 prevents accumulation of protein aggregates in cardiomyocytes

description: Protein homeostasis is crucial for maintaining cardiomyocyte (CM)
 function. Disruption of proteostasis results in accumulation of protein
 aggregates causing cardiac pathologies such as hypertrophy, dilated
 cardiomyopathy (DCM), and heart failure. Here, we identify the
 ubiquitin-specific peptidase 5 (USP5) as a critical determinant of protein
 quality control (PQC) in CM. CM-specific loss of mUsp5 leads to the
 accumulation of polyubiquitin chains and protein aggregates, cardiac
 remodeling, and eventually DCM. USP5 interacts with key components of the
 proteostasis machinery, including PSMD14 and absence of USP5 increases
 activity of the ubiquitin-proteasome system and autophagic flux in CMs.
 Cardiac-specific hUSP5 overexpression reduces pathological remodeling in
 pressure-overloaded mouse hearts and attenuates protein aggregate
 formation in titinopathy and desminopathy models. Since CMs from humans
 with end-stage DCM show lower USP5 levels and display accumulation of
 ubiquitinated protein aggregates, we hypothesize that therapeutically
 increased USP5 activity may reduce protein aggregates during DCM. Our
 findings demonstrate that USP5 is essential for ubiquitin turnover and
 proteostasis in mature CMs.
description: # Data from: The deubiquitinase USP5 prevents accumulation of protein
 aggregates in cardiomyocytes
 [https://doi.org/10.5061/dryad.1c59zw462](https://doi.org/10.5061/dryad.1c59zw462) ## Description of the data and file structure The Excel files contain the raw data used for statistical analysis and graph generation in the main and supplementary figures of the manuscript, 'The deubiquitinase USP5 prevents accumulation of protein aggregates in cardiomyocytes'. The methods used to generate the data are described in the manuscript. Each tab in the Excel sheet refers to an individual panel in the respective figures. ### Files and variables #### File: Eibach\_et\_al.\_R1\_Source\_Data\_Main\_Figures.xlsx **Description:**  Raw data for individual figure panels for different figures in the main manuscript.  **Source Data Main Figures** Data for main figures are summarized in a single excel file containing different tabs for different datasets. Figure 1D: This tab contains concentrations of biotinylated proteins identified in BirA* and USP5-BirA* (named as BioID-hUSP5long) transduced cells by using a BioID approach. Concentrations were determined by label-free quantification. Figure 2C: This tab contains relative values of USP5 protein levels in isolated cardiomyocytes from flox/flox, MCM and *Usp5* cKO hearts 15 days after the last tamoxifen administration. Figure 2F: This tab contains relative values of Ubiquitin protein levels in isolated cardiomyocytes from flox/flox, MCM and cKO hearts 15 days after the last tamoxifen administration. Figure 2H: This tab contains relative values of K48-Ubiquitin protein levels in isolated cardiomyocytes from flox/flox, MCM and cKO hearts 15 days after the last tamoxifen administration. Figure 2J: This tab contains relative values of Ubiquitin protein levels in primary non-cardiomyocytes (non-CMs), mouse embryonic fibroblasts (MEFs) and hepatocytes (hepatos) after performing Adenovirus-Null (Ad-Null) and Adenovirus-Cre (Ad-Cre) transduction. Figure 2I: This tab contains relative values of USP5 protein levels in primary non-cardiomyocytes (non-CMs), mouse embryonic fibroblasts (MEFs) and hepatocytes (hepatos) after performing Adenovirus-Null (Ad-Null) and Adenovirus-Cre (Ad-Cre) transduction. Figure 3A: This tab contains Relative Light Unit values for Chymotrypsin-(CT-L), Trypsin-(TL) and Caspase-(CL)-like activities in extracts of Wild type and *Usp5* cKO isolated cardiomyocytes, using catalytic-site specific substrates. Figure 3C: This tab contains relative values for Chymotrypsin-(CT-L) activity of different proteasome complexes (20S, 26S, 30S) in Wild Type and *Usp5* cKO cardiomyocytes 15 days after the last tamoxifen administration. Figure 3D: This tab contains ratios for Chymotrypsin-(CT-L) activity of different proteasome complexes (20S, 26S, 30S) in Wild Type and *Usp5* cKO cardiomyocytes 15 days after the last tamoxifen administration. Figure 3F: This tab contains relative values of 30S, 26S and 20S abundances in Wild Type and *Usp5* cKO cardiomyocytes 15 days after the last tamoxifen administration. Figure 3G: This tab contains ratios for 30S, 26S and 20S abundances in Wild Type and *Usp5* cKO cardiomyocytes 15 days after the last tamoxifen administration. Figure 3H: This tab contains values for specific activity calculated by ratio of relative Chymotrypsin activity and relative protein levels of the respective proteasome complexes (20S, 26S, 30S) in Wild Type and *Usp5* cKO cardiomyocytes 15 days after the last tamoxifen administration. Figure 3J: This tab contains mean values for number and size (µm) of protein aggregates quantified in isolated cardiomyocytes from flox/flox, MCM and *Usp5* cKO mice after the treatment with MG132. Figure 3K: This tab contains relative values for Chymotrypsin-(CT-L) activity after treatment with MG132 or DMSO in mouse embryonic fibroblasts (MEFs) and hepatocytes (hepatos). The cells were transduced with either Adenovirus-Null (Ad-Null) or Adenovirus-Cre (Ad-Cre). Figure 3M: This tab contains mean values for number and size (µm) of protein aggregates quantified after treatment with MG132 or DMSO in mouse embryonic fibroblasts (MEFs) and hepatocytes (hepatos). The cells were transduced with either Adenovirus-Null (Ad-Null) or Adenovirus-Cre (Ad-Cre). Figure 4B: This tab contains relative values of K63-Ubiquitin protein levels in isolated cardiomyocytes from flox/flox, MCM and cKO hearts 15 days after the last tamoxifen administration. Figure 4D: This tab contains relative values of p62 protein levels in isolated cardiomyocytes from flox/flox, MCM and cKO hearts 15 days after the last tamoxifen administration. Figure 4E: This tab contains relative values of LCI/II ratio in isolated cardiomyocytes from flox/flox, MCM and cKO hearts 15 days after the last tamoxifen administration. Figure 4G: This tab contains relative values of p62 protein levels after treatment with DMSO or Bafilomycon A1 in isolated cardiomyocytes from flox/flox, MCM and cKO hearts 15 days after the last tamoxifen administration. Figure 4H: This tab contains relative values of LCI/II ratio after treatment with DMSO or Bafilomycon A1 in isolated cardiomyocytes from flox/flox, MCM and cKO hearts 15 days after the last tamoxifen administration. Figure 4I: This tab contains relative values for LC3II flux assessed by subtracting relative LC3II protein levels in Bafilomycin A1-treated versus DMSO-treated cardiomyocytes. Cells were isolated from MCM, flox/flox, and *Usp5 *mice15 days after the last tamoxifen administration. Figure 4L: This tab contains ratio of cleaved Caspase-3 to total Caspase-3 in MCM, flox/flox, and *Usp5* cKO cardiomyocytes isolated 15 days after the last tamoxifen administration. Figure 5A: This tab contains survival rate of flox/flox, MCM and *Usp5* cKO mice monitored over a 35-day period following the administration of tamoxifen. Figure 5B: This tab contains physiological parameters body weight (BW), heart weight (HW), heart weight to body weight (HW:BW) and heart weight to tibia length (HW:TL) ratio in flox/flox, MCM and *Usp5* cKO mice 24 days after the last tamoxifen administration. Figure 5E: This tab contains values for myocardial wall thickness (µm) from the right ventricle (RV), intraventricular septum (IVS) and left ventricle (LV) of flox/flox, MCM and *Usp5* cKO hearts 24 days after the last tamoxifen administration. Figure 5H: This tab contains relative values of cardiac parameters ejection fraction (EF), cardiac output (CO) and heart beat in flox/flox, MCM and *Usp5* cKO mice 24 days after the last tamoxifen administration. Figure 5I: This tab contains percentage of fibrotic area of myocardial cross sections from control and *Usp5* cKO hearts isolated 24 days after the last tamoxifen administration. Figure 5J: This tab contains mean values of cardiomyocyte size (µm2) from flox/flox, MCM and *Usp5* cKO mice measured 15 days after the last tamoxifen administration. Figure 6B: This tab contains mean values for number and size (µm) of protein aggregates in hearts of control and dilated cardiomyopathy (DCM) patients. Figure 6D: This tab contains average percentage of cardiomyocytes containing Ubiquitin-positive aggregates and size (µm) of protein aggregates in hearts of control and dilated cardiomyopathy (DCM) patients. Figure 6F: This tab contains relative values of Ubiquitin protein levels in hearts of control and dilated cardiomyopathy (DCM) patients. Figure 6H: This tab contains average percentage of cardiomyocytes containing USP5-positive aggregates in hearts of control and dilated cardiomyopathy (DCM) patients. Figure 6J: This tab contains relative values of USP5 protein levels in hearts of control and dilated cardiomyopathy (DCM) patients. Figure 6L: This tab contains Relative values of short and long human USP5 in hearts of control and dilated cardiomyopathy (DCM) patients. Figure 6M: This tab contains relative values of human USP5 exon boundaries 6-7 and exon 15-16 in hearts of control and dilated cardiomyopathy (DCM) patients. Figure 7B: This tab contains relative values of USP5 protein levels in Cre and USP5 overexpressing (cOE) isolated cardiomyocytes. Figure 7D: This tab contains relative quantification values of Ubiquitin protein levels in Wild type (WT), Cre and USP5 overexpressing (cOE) isolated cardiomyocytes. Figure 7E: This tab contains relative values for expression of human USP5, murine Usp5 and stress markers in Cre and USP5 overexpressing (cOE) isolated cardiomyocytes. Figure 7F: This tab contains heart function parameters ventricular mass to body weight ratio and ventricular mass to tibia length in Cre and USP5 overexpressing (cOE) isolated cardiomyocytes. Figure 7G:  This tab contains heart function parameters left ventricular ejection fraction (LVEF) and diastolic left ventricular (LV) diameter in Cre and USP5 overexpressing (cOE) isolated cardiomyocytes. Figure 7H: This tab contains parameters for cardiac function monitored at different time points relative to surgery in control and USP5 overexpressing (cOE) mice. Data were used to produce Figure 7H, as well as Supplementary Figure 7B. Figure 7I: This tab contains parameters for cardiac function monitored at different time points relative to surgery in control and USP5 overexpressing (cOE) mice. Data were used to produce Figure 7H, as well as Supplementary Figure 7B. Figure 7K: This tab contains mean values for number and size (µm) of protein aggregates in control and USP5 overexpressing (cOE) cardiomyocytes at two different time points following surgery. Figure 7M: This tab contains relative values of Ubiquitin protein levels in control and USP5 overexpressing (cOE) isolated cardiomyocytes at two different time points following surgery. Figure 7O: This tab contains relative values of p62 protein levels and LCI/II ratio in control and USP5 overexpressing (cOE) isolated cardiomyocytes at two different time points following surgery. Figure 7P: This tab contains relative values for expression of fibrose marker genes in control USP5 overexpressing (cOE) isolated cardiomyocytes at two different time points following surgery. Figure 8C: This tab contains mean values for number and size (µm) of GFP-positive aggregates in Cre and USP5 overexpressing (cOE) cardiomyocytes after transduction with an adenovirus encoding wild-type titin (WT titin) and HMERF-linked mini-titins C31712R (CR), P30091L (PL) and P30091L + R32450W (PL+RW). Figure 8F: This tab contains mean values for number and size (µm) of GFP-positive aggregates in Cre and USP5 overexpressing (cOE) cardiomyocytes after transduction with an adenovirus encoding DCM-linked mini-titins p.Val22232Glu (VE), p.Gly27849Val (GV) and corresponding controls. Figure 9B: This tab contains relative values of USP5 protein levels in cardiomyocytes from Control-Desmin, DesR349P and DesR349P:USP5 mice. Figure 9D: This tab contains relative values of DESMIN protein levels in cardiomyocytes from Control-Desmin, DesR349P and DesR349P:USP5 mice. Figure 9F: This tab contains mean values for number of protein aggregates in cardiomyocytes from Control-Desmin, DesR349P and DesR349P:USP5 mice. Figure 9H: This tab contains relative values of Ubiquitin protein levels in cardiomyocytes from Control-Desmin, DesR349P and DesR349P:USP5 mice. Figure 9I: This tab contains relative values for Chymotrypsin-(CT-L) activity in cardiomyocytes from control, USP5 overexpressing (cOE) and R349P-Des:USP5 mice. Figure 9K: This tab contains relative values of p62 protein levels in cardiomyocytes from Control-Desmin, DesR349P and DesR349P:USP5 mice. Figure 9M: This tab contains relative values of LCI/II ratio in cardiomyocytes from Control-Desmin, DesR349P and DesR349P:USP5 mice. #### File: Eibach\_et\_al.\_R1\_Source\_Data\_Supplement.xlsx **Description:** Raw data for individual figure panels for different figures in the supplemental materials. **Source Data Supplementary Figures** Data for supplementary figures are summarized in a single excel file containing different tabs for different datasets.  Figure S2A: This tab contains log2 ratios of biotinylated proteins identified in triplicate experiments in BirA*-HA and USP5-BirA*-HA transduced HEK293 cells. Figure S2B: This tab contains log2 ratios and –log10 P-values of biotinylated proteins identified in triplicate experiments in BirA*-HA and USP5-BirA*-HA transduced HEK293 cells. Figure S2C: This tab contains log2 ratios of biotinylated proteins identified in triplicate experiments in Myc-BirA* and Myc-BirA*-USP5 transduced HEK293 cells. Figure S2D: This tab contains log2 ratios and –log10 P-values of biotinylated proteins identified in triplicate experiments in Myc-BirA* and Myc-BirA*-USP5 transduced HEK293 cells. Figure S2E: This tab contains -log10 P-values of GO terms of proteins biotinylated by USP5-BirA* with respect to biological processes. Figure S2F: This tab contains -log10 P-values of GO terms of proteins biotinylated by USP5-BirA* with respect to cellular components. Figure S3A: This tab contains relative values for mRNA expression of *Usp5* after treatment with MG132 or DMSO of flox/flox or *Usp5* cKO cardiomyocytes isolated 15 days after the last tamoxifen administration. Figure S3C: This tab contains -log10 P-values of GO terms (biological processes and cellular components) of differentially expressed genes in isolated cardiomyocytes from flox/flox and *Usp5* cKO hearts 15 days after the last tamoxifen administration. Figure S3D: This tab contains log2 ratios of differently expressed genes in cardiomyocytes from flox/flox and *Usp5* cKO hearts 15 days after the last tamoxifen administration. Figure S3E: This tab contains log2 ratios of differently expressed genes in cardiomyocytes from flox/flox and *Usp5* cKO hearts 15 days after the last tamoxifen administration. Figure S3F: This tab contains relative values for mRNA expression of *Nppa, Myh7/Myh6, Acta1, Fbxo32, Psmd4, and Uchl1* as well as polyubiquitin precursor genes *Ubb* and *Ubc* in flox/flox or *Usp5* cKO cardiomyocytes isolated 15 days after the last tamoxifen administration. Figure S4B: This tab contains P-values and log2 ratios showing significantly deregulated, selected ubiquitin-related proteins in *Usp5* cKO cardiomyocytes compared to controls. Figure S4C: This tab contains -log10 P-values of GO terms with respect to biological processes of differently expressed proteins in cardiomyocytes from flox/flox and cKO hearts 15 days after the last tamoxifen administration. Figure S4D: This tab contains -log10 P-values of GO terms with respect to cellular components of differentially expressed proteins in isolated cardiomyocytes from flox/flox and cKO hearts 15 days after the last tamoxifen administration. Figure S4F: This tab contains relative values for protein levels of 19S proteasome subunits in flox/flox, MCM and *Usp5*cKO cardiomyocytes isolated 15 days after the last tamoxifen administration. Figure S4H: This tab contains relative values for protein levels of 20S proteasome subunits in flox/flox, MCM and *Usp5*cKO cardiomyocytes isolated 15 days after the last tamoxifen administration. Figure S5D: This tab contains relative values for USP5 and LC3I/II ratio in HL-1 cardiomyocytes after transfection with siCTRL or siUSP5. Relative values for p62 protein levels in HL-1 cardiomyocytes after transfection with siCTRL or siUSP5 and treatment with MG132. Figure S5F: This tab contains mean values for number of protein aggregates in cardiomyocytes after transfection with siCTRL or siUSP5, and MG132 treatment. Figure S6B: This tab contains mean values for number of protein aggregates in cardiomyocytes from flox/flox, MCM and *Usp5* cKO mice isolated 15 days after the last tamoxifen administration. Figure S6C: This tab contains mean values for size (µm) of protein aggregates in cardiomyocytes from flox/flox, MCM and *Usp5* cKO mice isolated 15 days after the last tamoxifen administration. Figure S7A: This tab contains values for body weight monitored at different time points before and after surgery in control and USP5 overexpressing (cOE) mice. Figure S7B: This tab contains heart function parameters ventricular mass to body weight ratio and ventricular mass to tibia length at different time points before and after surgery in Cre and USP5 overexpressing (cOE) isolated cardiomyocytes. Figure S7C: This tab contains heart function parameters left ventricular ejection fraction (LVEF) and diastolic left ventricular (LV) diameter at different time points before and after surgery in Cre and USP5 overexpressing (cOE) isolated cardiomyocytes. ## Access information Other publicly accessible locations of the data: * none Data was derived from the following sources: * described in the manuscript 'The deubiquitinase USP5 prevents accumulation of protein aggregates in cardiomyocytes'