mitochondrial total protein content  (Bio-Rad)

Journal: bioRxiv

Article Title: Peroxisome protein import deficiency causes heart failure in mouse and human

doi: 10.1101/2025.10.13.681307

Figure Lengend Snippet: (A) Mitochondrial fragmentation in cPEX5 iKO iPSC-CMs observed by Mitotracker green staining. Structured illumination microscopy; Scale: 10 µm ( B-E ) Quantification of (A); Mitochondria analyzer ImageJ. Increased number of mitochondria per cell (B) with reduced number auf branches (C), decreased branch length (D) and decreased branch junctions per mitochondria (E). ( F ) TMRE (tetramethylrhodamin, 600nM) measurement shows reduced mitochondrial membrane potential in cPEX5 iKO . Normalized to MitoTracker green (MT). ( G-K ) Measurement of oxygen consumption rate (OCR, two-way ANOVA) in hiPSC-CMs (G) revealed no change in basal respiration (H) or ATP-linked OCR (I), but significant deficits in maximal respiration (J) and spare respiratory capacity (K) in cPEX5 iKO compared to WT cells. Normalized to cell number (nuclei). N=15 per group, Welch’s test. ( L ) ATP Assay in whole cell lysates. Normalized to WT, N=20 per group.

Article Snippet: The mitochondria pellet was washed and centrifuged again at 10000 g for 10 min. Mitochondrial total protein content was determined using the DC protein assay from BioRad according to manufacturer’s instructions.

Techniques: Staining, Microscopy, Membrane, ATP Assay

Journal: bioRxiv

Article Title: Peroxisome protein import deficiency causes heart failure in mouse and human

doi: 10.1101/2025.10.13.681307

Figure Lengend Snippet: (A) Cre-driven cardiac-specific Pex5 knockout (Pex5 cKO ). ( B,C ) IF of isolated cardiomyocytes shows peroxisomal ghosts (B, anti-PEX14) with increased area and deceased number of peroxisomes per cell and loss of ACAA1 (C) in peroxisomes. Scale: 10 µm. ( D ) Quantification of PEX14 staining (B). N = 30 cells, one-way ANOVA, Tukey’s multiple comparisons. ( F,G ) Confocal (upper row) and STED (lower row) microscope imaging of isolated cardiomyocytes from Pex5 cKO mice stained for PEX14 and CAV3 (F) or RYR2 (G) indicates increase in axial T-tubule structures (arrowheads). Arrows indicate peroxisomal lumen in WT cardiomyocytes, which is not seen in Pex5 cKO cardiomyocytes. Scale: 5 µm. ( H,I ) Analysis of peroxisomes in confocal and STED images (F,G). Solidity of peroxisomes calculated as ratio of the area and convex area reveals that peroxisomes in Pex5 cKO are more solid (H). This can be seen with STED, but not confocal microscopy. Roundness of peroxisomes is calculated based on the area and major axis. Peroxisomes in Pex5 cKO are more round compared to WT (I). STED is a more sensitive approach than confocal when imaging less round objects. N = 3 mice, one-way ANOVA, Tukey’s multiple comparisons. ( J-M ) Loss of DAP-AT (J) and PHYH (L) in peroxisomes of isolated cardiomyocytes of Pex5 cKO mice. Scale: 10 µm. (K,M) Quantification of J,L (Manders coefficient). M1= Fraction of PEX14 / PMP70 that overlaps with PHYH or DAP-AT, M2= Fraction of PHYH or DAP-AT that overlaps with PEX14/PMP70. N = 5 - 9 cells per group, one-way ANOVA, Tukey’s multiple comparisons. ( N ) Reduction of Gnpat and significant downregulation of Phyh mRNA. qPCR. One-way ANOVA, Tukey’s multiple comparisons. N=3 mice per group. Normalized to Gapdh . ( O ) Loss of C18 plasmalogens in Pex5 cKO hearts. N = 3 hearts per group, one-way ANOVA, Tukey’s multiple comparisons. ( P ) EM images of heart tissue sections show mitochondrial fragmentation and sarcomere loss in Pex5 cKO hearts. Scale: 500 nm. ( Q-T ) Quantification of (P). N = 3 mice per group, one-way ANOVA, Tukey’s multiple comparisons.

Article Snippet: The mitochondria pellet was washed and centrifuged again at 10000 g for 10 min. Mitochondrial total protein content was determined using the DC protein assay from BioRad according to manufacturer’s instructions.

Techniques: Knock-Out, Isolation, Staining, Microscopy, Imaging, Confocal Microscopy

Journal: bioRxiv

Article Title: Peroxisome protein import deficiency causes heart failure in mouse and human

doi: 10.1101/2025.10.13.681307

Figure Lengend Snippet: ( A-D ) Electrophysiological characterization of cardiac electrograms. ( A ) Original cardiac electrogram (ECG) recording of explanted hearts from a WT heart (black) and a Pex5 cKO heart (red). (B-D) Statistical analysis of ECG parameters during spontaneous beating. QRS duration, 12.87 ± 0.12 vs. 20.20 ± 0.22 ms (B), QT interval, 48.2 ± 0.4 ms vs. 73.6 ms ± 0.5 ms (C) and ventricular effective refractory period (ERP), 38.9 ± 1.9 vs. 65.4 ± 5.0 ms (D) in WT and Pex5 cKO hearts. N = 14 - 18 hearts per group. ( E-G ) Autofluorescence of NAD(P)H and FAD were measured in murine cardiomyocytes exposed to the indicated protocol (top). Level of oxidized FAD is reduced compared to WT and did not show an increase following workload transition (E, F); Pre = basal FAD level at 0,5 Hz stimulation; Post = 40-60 s after initiation of 5 Hz stimulation. (G) NADH oxidation following workload transition is slightly reduced in cKO compared to WT cells and the NAD(P)H redox state after the initial oxidation cannot be maintained throughout the stress protocol in Pex5 cKO compared to WT cells. N = 6-21 cells from 3 mice per group. ( H, I ) Cardiomyocytes were loaded with TMRM to measure changes in the mitochondrial membrane potential (ΔΨ m ). Total membrane potential is significantly decreased in Pex5 cKO cells (H). Pex5 cKO cells show a more stable membrane potential under the stress protocol than WT cells (I, traces normalized at t=0). N = 40 cells from 3 mice per group. (E-I) Two-way ANOVA, Bonferroni’s multiple comparisons. ( J-M ) In isolated myocytes force development was determined by increasing the stimulation frequency from 1 to 4 Hz for 60 s and back after stepwise stretching the cell until the diastolic sarcomere length reached 2.0 µm. Example from WT (J). Pex5 cKO cells show less force development (K) compared to WT myocytes. Contraction velocity (L) and relaxation velocity (M) are significantly decreased in Pex5 cKO cells. WO = washout, BL = baseline. 6-9 cells from 3-4 mice per group, two-way ANOVA, Bonferroni’s multiple comparisons.

Article Snippet: The mitochondria pellet was washed and centrifuged again at 10000 g for 10 min. Mitochondrial total protein content was determined using the DC protein assay from BioRad according to manufacturer’s instructions.

Techniques: Membrane, Isolation

Journal: bioRxiv

Article Title: Peroxisome protein import deficiency causes heart failure in mouse and human

doi: 10.1101/2025.10.13.681307

Figure Lengend Snippet: Interorganellar crosstalk involving peroxisomes is a prerequisite for normal cardiac physiology in healthy cardiomyocytes. Loss of functional peroxisomes leads to mitochondrial aberrations with reduced respiration and disturbs SR Ca 2+ homeostasis and T-tubule morphology. The disrupted organelle function results in irregular conduction, decreased contractility and force generation on cellular level, eventually leading to systolic and diastolic dysfunction, and finally failure of the whole organ. Vice versa, cardiac hypertrophy and heart failure trigger peroxisome responses. (Designed with BioRender.com ).

Article Snippet: The mitochondria pellet was washed and centrifuged again at 10000 g for 10 min. Mitochondrial total protein content was determined using the DC protein assay from BioRad according to manufacturer’s instructions.

Techniques: Functional Assay