PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

Figures

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  Figure 1. PAD2 protein is increased in trauma patients with hemorrhagic shock and knockout of Pad2 improves hemorrhagic ventricular arrhythmias. (A) Peripheral blood plasma PAD2 concentration (ELISA) in patients with traumatic hemorrhagic shock (n = 22) and healthy volunteers (n = 30),****p < 0.0001; (B) Correlation analysis between PAD2 concentration and lactate concentration in peripheral blood plasma of patients with traumatic hemorrhagic shock (n = 22), r = 0.5174, ***p = 0.0002; (C) Expression of PAD2 in RNA Seq of cardiac tissue in GEO datasets of patients with arrhythmia compared to healthy volunteers; (D) Construction strategy of Pad2 knockout (Pad2−/−) mice (upper), Pad2 expression level in heart tissues of Pad2−/− mice and WT mice by western blot (bottom); (E) Schematic diagram of hemorrhagic shock model; (F) After hemorrhagic shock, the Kaplan-Meier survival rate curve of WT mice (n = 7) and Pad2−/− mice (n = 7), ****p < 0.0001; (G) Representative electrocardiogram monitoring images of WT mice and Pad2−/− mice in hemorrhagic shock model; (H) The statistical data of heart rate of WT mice before and after hemorrhagic shock (n = 6, ***p = 0.0008), and the statistical data of heart…

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  Figure 2. Cardiac overexpression of PAD2 increases mortality and susceptibility to ventricular arrhythmias following hemorrhagic shock. Construct cardiomyocyte PAD2 overexpression in WT mice by inject AAV9-PAD2 via tail vein of WT for 4 weeks, AAV9-MCS injection as control; Construct cardiomyocyte PAD2 reintroduction in Pad2−/− mice by inject AAV9-PAD2 via tail vein of Pad2−/− mice for 4 weeks, AAV9-MCS injection as control; (A) Construction strategy of cardiomyocyte PAD2 overexpression in WT mice, cardiomyocytes PAD2 reintroduction in Pad2−/− mice; (B) Representative images of confocal microscopy to verify the overexpression (top)/reintroduction (bottom) efficiency of Pad2 in WT/ Pad2−/− mouse hearts; (C) Representative protein immunoblotting images to validate the overexpression (top)/reintroduction (bottom) efficiency of Pad2 in WT/ Pad2−/− mouse hearts; (D) Kaplan-Meier survival rate curve of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice after hemorrhagic shock, n = 6 vs. n = 5, **p = 0.0044; (E) Representative electrocardiogram monitoring images of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice after hemorrhagic shock in…

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  Figure 3. PAD2 deficiency protects cardiomyocytes from arrhythmia by maintaining Ca2+ homeostasis. (A) Representative diagram of cytosolic Ca2+ recordings of adult mouse cardiomyocytes in response to 10-V, 0.5-Hz to 7.0-Hz electronic pacing; n = 20 from 5 mice in WT group, n = 18 from 5 mice in Pad2 KO group. (B) Average Ca2+ decay Tau of adult mouse cardiomyocytes in response to 10-V, 0.5-Hz (n = 6 vs. n = 6, **p = 0.0025), 2.0-Hz (n = 6 vs. n = 6, **p = 0.0015), and 3.0 Hz (n = 4 vs. n = 6, ***p = 0.0005) separately. (C) Representative confocal images of cytosolic Ca2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult mouse cardiomyocytes, (D) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F0) over time. (E) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F0, and n = 10 cells from 6 mice in each group. **p < 0.0001. (F) Representative confocal images of cytosolic Ca2+ via the Fluo-4 AM fluorescence signal in response to 10…

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  Figure 4. Hypoxia induces PAD2 translocation and increases its co-localization with the SR. (A) Representative Western blot (left) and average data (right) illustrating Pad2 protein levels in cardiomyocytes under normoxic or hypoxic conditions for 1 hour. Each group consists of n = 6 vs. n = 6 samples. (B) Immunofluorescence staining (top) showing the translocation of Pad2 out of the nucleus under hypoxic stimulation. Scale bar = 5 μm (upper)Scale bar = 10 μm (bottom). The quantification of Pad2 fluorescence intensity in the nucleus and cytoplasm is shown below, with values expressed as the ratio of average fluorescence intensity to pixels. After hypoxia stimulation, the average fluorescence intensity of Pad2 in the nucleus decreased significantly (n = 7 vs. n = 7, *p = 0.013), while the average fluorescence intensity of Pad2 in the cytoplasm increased significantly (n = 7 vs. n = 7, **p = 0.0078). H9c2 cells were exposed to either normoxia or hypoxia, and cytoplasmic and nuclear proteins were isolated. (C) Quantification of Pad2 protein levels in the cytoplasm and nucleus was performed using Western blotting (WB). Representative Western blot (top) and average data (bottom) demonstrate a…

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  Figure 5. Hypoxia inhibits SERCA2a activity through PAD2-mediated citrullination. Neonatal rat cardiomyocytes were infected with Ad-PAD2, and CoIP-MS (co-immunoprecipitation mass spectrometry) was performed using Pad2 and IgG controls separately. (A) Heatmap of CoIP-MS. (B) Gene Ontology (GO) enrichment analysis of CoIP-MS. (C) KEGG pathway enrichment analysis of CoIP-MS. (D) ER Proteins interacting with Pad2. (E) The results of protein immunoprecipitation (CoIP) showed that endogenous Pad2 protein interacts with SERCA2a protein in neonatal rat cardiomyocytes. (F) Western blot analysis (left) and average data (right) showing SERCA2a protein levels in neonatal rat cardiomyocytes under normoxic or hypoxic conditions (n = 4 vs. n = 4 per group). (G) Measurement of Pad2 enzymatic activity in neonatal rat cardiomyocytes followed by normoxia or hypoxia stimulation for 1 hour. (n = 6 for each group). (H) Measurement of SERCA2a enzymatic activity in neonatal rat cardiomyocytes infected with Ad-LacZ or Ad-PAD2 for 48 hours, followed by normoxia or hypoxia stimulation for 1 hour (n = 7 vs. n = 7 per group, *p = 0.004, *p = 0.0478, ****p < 0.0001). (I) Measurement of SERCA2a enzymatic activity in…

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  Figure 6. PAD2 inhibitor prevents hemorrhagic shock-induced cardiac arrhythmia. Thirty minutes before the hemorrhagic shock model, mice were treated with different concentrations of the Pad2 selective inhibitor AFM-30a (10 mg/kg, 20 mg/kg, and 40 mg/kg) via intraperitoneal injection, with DMSO as the vehicle control. (A) Survival rates of hemorrhagic shock mice treated with various concentrations of AFM-30a. n = 12 for each treated group and n = 16 for the vehicle group. *p = 0.0338, **p = 0.0084. (B) Serum lactate concentrations from mice treated with various concentrations of AFM-30a. n = 6 for each treated group and n = 10 for the vehicle group. *p = 0.0251, *p = 0.0323. (C) Representative electrocardiogram (ECG) monitoring images of mice treated with vehicle (DMSO), or 40 mg/kg of AFM-30a for 30 minutes before the hemorrhagic shock model. In hemorrhagic shock, (D) Statistical values of heart rate from ECG recordings (top). Statistical values of QT interval from ECG recordings (bottom). n = 6 for each group. (E) Representative diagram of cytosolic Ca2+ recordings of adult mouse cardiomyocytes pretreated with 25 μM of AFM-30a or DMSO for 24 hours in response to 10-V, 0.5-Hz to 7.0-Hz…