Peroxiredoxin-6 protects against oxidative stress-mediated hypertrophic remodeling in JPH2-A399S knock-in mice

Figures

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  Fig. 1. JPH2 interacts with PRDX6, which is downregulated in the IVS of JPH2-A399S mice and in human failing hearts. (A) Volcano plot of IP enrichment vs statistical significance from JPH2 co-immunoprecipitation mass spectrometry showing PRDX6 as one of the most abundant proteins in the JPH2 pulldown. RyR2 is shown as a positive control. The x-axis shows log2(IP enrichment), where IP represents the immunoprecipitation enrichment ratio. The y-axis shows -log10(p) using nominal p-values. Horizontal dashed line at -log10(0.05) = 1.3 indicates threshold corresponding to p < 0.05 (B) Co-immunoprecipitation immunoblot confirming the physical interaction between JPH2 and PRDX6. (C, D) Representative immunoblot (C) and quantification (D) of PRDX6 protein levels in the interventricular septum (IVS) and left ventricular free wall (LVFW) of wild-type (WT) and JPH2-A399S knock-in mice. PRDX6 abundance is selectively reduced in the IVS of A399S mice. N = 4–5 mice per group. Data analyzed by two-way ANOVA with Fisher's LSD post-hoc test. (E, F) Representative immunoblot (E) and quantification (F) of PRDX6 protein levels in left ventricular tissue from patients with ischemic cardiomyopathy (ICM)…

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  Fig. 2. Nanoscale colocalization of JPH2 with PRDX6 and preserved JPH2-RyR2 organization in JPH2-A399S IVS cardiomyocytes. Both confocal and stimulated emission depletion (STED) super-resolution microscopy were used to examine the spatial relationship of JPH2 (red) with either PRDX6 (green; A-D) or RyR2 (green; E-H) in isolated IVS cardiomyocytes from WT and JPH2-A399S mice. Representative confocal images are shown next to corresponding STED images for the same region of interest. White boxes (B, F) indicate regions magnified in rightward images (C, G). White arrows highlight the diagonal line's direction used for signal intensity profiling. Line-scan intensity profiles illustrate the co-immunofluorescence intensity distributions, confirming the nanometric spatial association between the labeled protein pairs, each for JPH2/PRDX6 (D) and JPH2/RyR2 (H). Scale bars: 10 μm (confocal overview; A, E) and 1 μm (magnified confocal and corresponding STED panels B, F).

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  Fig. 3. JPH2-A399S mice exhibit increased oxidative stress in the IVS. (A, B) Validation of the dihydroethidium (DHE) probe in WT cardiomyocytes. Representative confocal images (A) and quantification (B) of DHE fluorescence in cells treated with vehicle (no DHE), 20 μM DHE, or 20 μM DHE + 300 μM H2O2. n = 4–8 cells. (C, D) Representative DHE fluorescence images (C) and quantification of the IVS-to-LVFW fluorescence ratio (D) in freshly isolated cardiomyocytes from WT and JPH2-A399S mice. N = 6–7 mice per group and data in (D) was analyzed by Mann-Whitney U test.

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  Fig. 4. AAV9-mediated overexpression of PRDX6 rescues IVS hypertrophy in JPH2-A399S mice. (A) Schematic of the AAV9-TnT4-PRDX6-emGFP vector used for cardiac myocyte-specific overexpression of PRDX6. (B) Representative long-axis echocardiographic images from WT, JPH2-A399S, and JPH2-A399S mice treated with AAV9-PRDX6. Red bars indicate the interventricular septum (IVS). (C, D) Quantification of interventricular septal thickness in diastole (IVS;d) (C) and left ventricular ejection fraction (EF) (D). N = 3–4 mice per group. Data analyzed by one-way ANOVA.

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  Fig. 5. Proposed model: PRDX6 downregulation and oxidative stress link the JPH2-A399S variant to region-specific hypertrophic cardiomyopathy. Schematic illustration summarizing the key findings of the study. The JPH2-A399S mutation leads to selective reduction of PRDX6 in the IVS, resulting in increased ROS production, which drive IVS-specific hypertrophy. AAV9-mediated restoration of PRDX6 rescues the hypertrophic phenotype. Figure made using BioRender.