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Proteintech anti pad2
Anti Pad2, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+pad2/PADI2+Antibody/pmc12966709-517-28-29
Average 94 stars, based on 74 article reviews

anti pad2 - by Bioz Stars, 2026-09

94/100 stars

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Produced:

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis
Article Snippet: The cells were immunostained using anti-PAD2 (1:200, 12110-1-AP, Proteintech) and anti-PAD4 (1:200, 17373-1-AP, Proteintech). .. Anti-mouse SAA1/2 (1:100, ab199030, Abcam), anti-citrulline (1:200, ab100932, Abcam), anti-citrulline (1:200, MA5-27573, Thermo) anti-CCP Polyclonal (1:200, bs-1053R, Bioss), anti-mouse CD45 (1:200, 103101, BioLegend), anti-mouse CD11b (1:200, 101202, BioLegend), and rat anti-mouse MECA32 (1:200, 550563, BD Pharmingen), anti-Fbg (1:200, A0080, Dako), anti-PAD2 (1:300, Proteintech), anti-PAD4 (1:300, Proteintech), and anti-citrullinated Fbg clone A-s1 (1:100, produced in our laboratory), were used for immunostaining the frozen mice lung tissue sections. .. We also used anti-mouse SAA3 antibody (1:100, produced in our laboratory) and Alexa Fluor secondary antibodies (1:400, A21208, A21207, A21447, A21202, A21203, A21206, Thermo, and ab150111, ab150075, ab150156, ab150155, Abcam) for signal visualization during confocal microscopy.

Immunostaining:

Saline:

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response
Article Snippet: Briefly, equal amounts of cell extracts were separated by SDS-PAGE and transferred to Immobilon-P membranes (Merck Millipore). .. These were blocked in Tris buffered saline (TBS) solution containing 0.05% Tween 20 and 5% milk and then incubated overnight at 4°C with appropriate primary antibodies: anti-CCP (Biorbyt, orb10262), anti-PAD2 (Proteintech, 12110-1-AP), anti-PAD4 (Proteintech,17373-1-AP), anti-actin (Sigma-Aldrich, A2066), anti-Nucleoprotein(N)_SARS-CoV-2 (GeneTex, 3851), anti-Spike(S)_SARS-CoV-2 (GeneTex, 1A9). .. Membranes were then washed and incubated for 2 h at room temperature with HRP- Jo urn al Pr e-p roo f 29 conjugated secondary antibodies (ECL anti-rabbit/mouse IgG, Cytiva).

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response
Article Snippet: Briefly, equal amounts of cell extracts were separated by SDS-PAGE and transferred to Immobilon-P membranes (Merck Millipore). .. These were blocked in Tris-buffered saline (TBS) solution containing 0.05% Tween 20 and 5% milk and then incubated overnight at 4°C with appropriate primary antibodies: anti-CCP (Biorbyt, orb10262), anti-PAD2 (Proteintech, 12110-1-AP), anti-PAD4 (Proteintech,17373-1-AP), anti-actin (Sigma-Aldrich, A2066), anti-Nucleoprotein(N)_SARS-CoV-2 (GeneTex, 3851), anti-Spike(S)_SARS-CoV-2 (GeneTex, 1A9). .. Membranes were then washed and incubated for 2 h at room temperature with HRP-conjugated secondary antibodies (ECL anti-rabbit/mouse IgG, Cytiva).

Incubation:

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response
Article Snippet: Briefly, equal amounts of cell extracts were separated by SDS-PAGE and transferred to Immobilon-P membranes (Merck Millipore). .. These were blocked in Tris-buffered saline (TBS) solution containing 0.05% Tween 20 and 5% milk and then incubated overnight at 4°C with appropriate primary antibodies: anti-CCP (Biorbyt, orb10262), anti-PAD2 (Proteintech, 12110-1-AP), anti-PAD4 (Proteintech,17373-1-AP), anti-actin (Sigma-Aldrich, A2066), anti-Nucleoprotein(N)_SARS-CoV-2 (GeneTex, 3851), anti-Spike(S)_SARS-CoV-2 (GeneTex, 1A9). .. Membranes were then washed and incubated for 2 h at room temperature with HRP-conjugated secondary antibodies (ECL anti-rabbit/mouse IgG, Cytiva).

Proximity Ligation Assay:

Article Title: Small molecule activates citrullination through targeting PAD2.
Article Snippet: Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, and Center for Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China School of pharmacy, Fudan University, Shanghai 201203, People’s Republic of China Department of Pharmacy, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, People’s Republic of China

Staining:

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis
Article Snippet: .. The specimens were embedded in OCT compound and cut, then slides were stained using anti-PAD2 (0.5 mg/mL, Proteintech) and anti-PAD4 (0.5 mg/mL, Proteintech) antibodies and control IgG (0.5 mg/mL, 3900 S, Cell Signaling Technology). .. Signals were examined by a fluorescent microscope (BZ-9000, Keyence, Osaka, Japan).

Control:

Image Search Results

$a Immunofluorescence analysis showing efficient uptake of Alexa Fluor 488-labeled CitH3 (5 µg/mL) by wild-type (WT) BMDMs but not by Tlr2⁻/⁻ BMDMs. Cellular intensity of Alexa-488 was quantified ( n = 5). Statistical analysis was performed using two-sided, Unpaired t test with Welch’s correction. b Cytokine profiling of pro- and anti-inflammatory mediators (IL-6, IL-1β, IL-10, TNFα, IFN-α, IFN-β) in supernatants of WT and Tlr2⁻/⁻ BMDMs treated with H3 or CitH3 peptides (15 µg/mL) for 16 h ( n = per group). LPS (200 ng/mL) served as positive control. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. c Immunocytochemistry demonstrating nuclear translocation of PAD2 in BMDMs after 1 h exposure to CitH3 peptide (15 µg/mL). LPS (200 ng/mL) served as a positive control. PAD2 was stained with Alexa Fluor 488 (green), and nuclei were counterstained with DAPI (blue). Quantification of PAD2 nuclear localization was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 3 per group). d Subcellular fractionation analysis confirming significant nuclear localization and citrullination of PAD2 in WT BMDMs following treatment with H3, CitH3, or LPS. Quantification was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test ( n = per group). e In vitro citrullination assay showing MPB-tagged PAD2-mediated citrullination of H3 in a Ca²⁺-dependent manner. EGTA-mediated Ca²⁺ chelation eliminated CitH3 production. Similar results from three independent replicates. f In vitro citrullination assay showing PAD2 citrullination. PAD2 auto-citrullination was confirmed as His-tagged recombinant PAD2 co-incubated with MBP-tagged PAD2 resulted in PAD2 citrullination. PAD4 also promotes PAD2 citrullination. Quantification was performed by Image J, citrullination signals were normalized to corresponding input signals and further normalized to the matching EGTA-treated group in each replicate experiments. Statistical analysis was performed using two-sided t-test.Data are presented as mean ± SD. Statistical significance in all panels was determined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.$

Journal: Nature Communications

Article Title: A citrullinated histone H3 monoclonal antibody for immune modulation in sepsis

doi: 10.1038/s41467-025-62788-6

Figure Lengend Snippet: a Immunofluorescence analysis showing efficient uptake of Alexa Fluor 488-labeled CitH3 (5 µg/mL) by wild-type (WT) BMDMs but not by Tlr2⁻/⁻ BMDMs. Cellular intensity of Alexa-488 was quantified ( n = 5). Statistical analysis was performed using two-sided, Unpaired t test with Welch’s correction. b Cytokine profiling of pro- and anti-inflammatory mediators (IL-6, IL-1β, IL-10, TNFα, IFN-α, IFN-β) in supernatants of WT and Tlr2⁻/⁻ BMDMs treated with H3 or CitH3 peptides (15 µg/mL) for 16 h ( n = per group). LPS (200 ng/mL) served as positive control. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. c Immunocytochemistry demonstrating nuclear translocation of PAD2 in BMDMs after 1 h exposure to CitH3 peptide (15 µg/mL). LPS (200 ng/mL) served as a positive control. PAD2 was stained with Alexa Fluor 488 (green), and nuclei were counterstained with DAPI (blue). Quantification of PAD2 nuclear localization was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 3 per group). d Subcellular fractionation analysis confirming significant nuclear localization and citrullination of PAD2 in WT BMDMs following treatment with H3, CitH3, or LPS. Quantification was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test ( n = per group). e In vitro citrullination assay showing MPB-tagged PAD2-mediated citrullination of H3 in a Ca²⁺-dependent manner. EGTA-mediated Ca²⁺ chelation eliminated CitH3 production. Similar results from three independent replicates. f In vitro citrullination assay showing PAD2 citrullination. PAD2 auto-citrullination was confirmed as His-tagged recombinant PAD2 co-incubated with MBP-tagged PAD2 resulted in PAD2 citrullination. PAD4 also promotes PAD2 citrullination. Quantification was performed by Image J, citrullination signals were normalized to corresponding input signals and further normalized to the matching EGTA-treated group in each replicate experiments. Statistical analysis was performed using two-sided t-test.Data are presented as mean ± SD. Statistical significance in all panels was determined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used in immunocytochemistry assays: PAD2 (12110-1-AP), PAD4 (17373-1-AP), Rab5 (CST, 3547), EEA1 (CST, 3288), MPO (Abcam ab208670)

Techniques: Immunofluorescence, Labeling, Positive Control, Immunocytochemistry, Translocation Assay, Staining, Fractionation, In Vitro, Recombinant, Incubation

a LDH release from supernatants of BMDMs treated with increasing doses of CitH3 or unmodified H3 peptides for 24 h at 37 °C, showing CitH3-induced cytotoxicity ( n = per group). b Cell viability, assessed using the CCK8 assay, demonstrated significant reduction following CitH3 peptide treatment compared to the H3 peptide ( n = 5 per group). c BMDMs treated with 50 µg/mL CitH3 peptide and increasing doses of hCitH3-mAb or human IgG. LDH release was measured to evaluate cytotoxicity ( n = per group). d , Viability of BMDMs treated as in ( c ) was measured using the CCK8 assay. hCitH3-mAb significantly preserved cell viability compared to control IgG ( n = per group). e Immunoblot analysis of CitH3 in BMDMs treated with 15 μg/mL CitH3 or H3 peptides. Cells were washed three times prior to collection. And given that the CitH3 peptide is only ~30 amino acids in length, the bands observed at ~17 kDa are interpreted as endogenous cellular CitH3 protein detected at various time points. Similar results from three independent replicates. f Western blot analysis of citrullinated proteins in BMDMs from WT, Pad2⁻/⁻, and Pad2/4⁻/⁻ mice following 1 h treatment with 15 μg/mL CitH3 peptide. CitH3-induced citrullination was PAD2-dependent. Similar results from four independent replicates. g Quantification of IL-6, IL-1β, TNFα, IFN-α, and IFN-β levels in the supernatants of BMDMs treated with 25 μg/mL CitH3 peptide for 24 h ( n = 6 per group). hCitH3-mAb effectively reduced CitH3-induced cytokine elevation in a dose-dependent manner, whereas control IgG had no effect. Data are presented as mean ± SD. Statistical analysis: One-way ANOVA followed by Dunnett’s multiple comparisons test was applied throughout. Comparisons were made to the ‘0’ group in ( a –d ), and the CitH3-treated group in ( g ), as indicated. Significance thresholds: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: A citrullinated histone H3 monoclonal antibody for immune modulation in sepsis

doi: 10.1038/s41467-025-62788-6

Figure Lengend Snippet: a LDH release from supernatants of BMDMs treated with increasing doses of CitH3 or unmodified H3 peptides for 24 h at 37 °C, showing CitH3-induced cytotoxicity ( n = per group). b Cell viability, assessed using the CCK8 assay, demonstrated significant reduction following CitH3 peptide treatment compared to the H3 peptide ( n = 5 per group). c BMDMs treated with 50 µg/mL CitH3 peptide and increasing doses of hCitH3-mAb or human IgG. LDH release was measured to evaluate cytotoxicity ( n = per group). d , Viability of BMDMs treated as in ( c ) was measured using the CCK8 assay. hCitH3-mAb significantly preserved cell viability compared to control IgG ( n = per group). e Immunoblot analysis of CitH3 in BMDMs treated with 15 μg/mL CitH3 or H3 peptides. Cells were washed three times prior to collection. And given that the CitH3 peptide is only ~30 amino acids in length, the bands observed at ~17 kDa are interpreted as endogenous cellular CitH3 protein detected at various time points. Similar results from three independent replicates. f Western blot analysis of citrullinated proteins in BMDMs from WT, Pad2⁻/⁻, and Pad2/4⁻/⁻ mice following 1 h treatment with 15 μg/mL CitH3 peptide. CitH3-induced citrullination was PAD2-dependent. Similar results from four independent replicates. g Quantification of IL-6, IL-1β, TNFα, IFN-α, and IFN-β levels in the supernatants of BMDMs treated with 25 μg/mL CitH3 peptide for 24 h ( n = 6 per group). hCitH3-mAb effectively reduced CitH3-induced cytokine elevation in a dose-dependent manner, whereas control IgG had no effect. Data are presented as mean ± SD. Statistical analysis: One-way ANOVA followed by Dunnett’s multiple comparisons test was applied throughout. Comparisons were made to the ‘0’ group in ( a –d ), and the CitH3-treated group in ( g ), as indicated. Significance thresholds: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used in immunocytochemistry assays: PAD2 (12110-1-AP), PAD4 (17373-1-AP), Rab5 (CST, 3547), EEA1 (CST, 3288), MPO (Abcam ab208670)

Techniques: CCK-8 Assay, Control, Western Blot

Effect of AMF30a on in vitro proliferation of human corneal endothelial cells (hCECs). AMF30a (5µM, PAD2 inhibitor, Cayman, Ann Arbor, Michigan) was applied for 48 h. ( A ) hCECs were identified using immunofluorescence staining of ZO1 and CD166. ( B - C ) Cell area of hCECs were evaluated using cell morphological pictures. Scale bar = 100 μm. ( D and E ) Cell proliferation was measured using CCK-8 cell viability assay and BrdU incorporation assay. ( F ) Cytotoxicity was evaluated using LDH cytotoxicity assay (1.002 ± 0.047 vs. 0.925 ± 0.018). ( G - I ) Cell cycle analysis was assessed using PI staining. (J and K) Wound healing assay was performed using the measurement of scratched ares. Scale bar = 500 μm. ( L and N ) Intracellular oxidative stress levels were measured using DCF-DA assay. Scale bar = 50 μm. * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of AMF30a on in vitro proliferation of human corneal endothelial cells (hCECs). AMF30a (5µM, PAD2 inhibitor, Cayman, Ann Arbor, Michigan) was applied for 48 h. ( A ) hCECs were identified using immunofluorescence staining of ZO1 and CD166. ( B - C ) Cell area of hCECs were evaluated using cell morphological pictures. Scale bar = 100 μm. ( D and E ) Cell proliferation was measured using CCK-8 cell viability assay and BrdU incorporation assay. ( F ) Cytotoxicity was evaluated using LDH cytotoxicity assay (1.002 ± 0.047 vs. 0.925 ± 0.018). ( G - I ) Cell cycle analysis was assessed using PI staining. (J and K) Wound healing assay was performed using the measurement of scratched ares. Scale bar = 500 μm. ( L and N ) Intracellular oxidative stress levels were measured using DCF-DA assay. Scale bar = 50 μm. * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Article Snippet: Primary antibodies were as follows: rabbit anti-human CD166 antibody (ab109215, Abcam, 1∶500 dilution), rabbit anti-human ZO-1 antibody (sc-10804, Santa Cruz, 1∶500 dilution), mouse anti-human ERK1/2 antibody (sc-514032, Santa Cruz, 1∶500 dilution); mouse anti-human pERK1/2 antibody (sc-136521, Santa Cruz, 1∶500 dilution); mouse anti-human YAP antibody (sc-376830, 1∶500 dilution); rabbit anti-pYAP antibody (PA5-17481, Invitrogen, 1∶500 dilution); rabbit anti-human PAD2 antibody (12110-1-AP, Proteintech, 1∶500 dilution); mouse anti-ATF4 antibody (sc-390063, 1∶500 dilution); mouse anti-human ROCK1 antibody (sc-17794, 1∶500 dilution); mouse anti-ROCK2 antibody (sc-398519, 1∶500 dilution); or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1∶5000 dilution).

Techniques: In Vitro, Immunofluorescence, Staining, CCK-8 Assay, Viability Assay, BrdU Incorporation Assay, LDH Cytotoxicity Assay, Cell Cycle Assay, Wound Healing Assay

Effect of cytokine on human corneal endothelial cells. ( A ) Immunofluorescence staining of PAD2 shows the distribution of PAD2. Scale bar = 100 μm. ( B and C ) Western blot of PAD2 was used to evaluate the PAD2 levels. ( D and E ) ATF4 levels were evaluated using western blot. ( F ) Immunofluorescence staining of peptidyl-citrulline shows the intensity and distribution of peptidyl-citrulline. Scale bar = 100 μm. * p < 0.05.

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of cytokine on human corneal endothelial cells. ( A ) Immunofluorescence staining of PAD2 shows the distribution of PAD2. Scale bar = 100 μm. ( B and C ) Western blot of PAD2 was used to evaluate the PAD2 levels. ( D and E ) ATF4 levels were evaluated using western blot. ( F ) Immunofluorescence staining of peptidyl-citrulline shows the intensity and distribution of peptidyl-citrulline. Scale bar = 100 μm. * p < 0.05.

Techniques: Immunofluorescence, Staining, Western Blot

Schematic diagram summarizing PAD2-mediated signaling in hCECs.

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Schematic diagram summarizing PAD2-mediated signaling in hCECs.

Techniques:

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Article Snippet: The cells were treated overnight with either mouse anti-E-cadherin antibody (sc-8426; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-YAP antibody (sc-376830; Santa Cruz Biotechnology), rabbit anti-PAD2 antibody (sc-293271; Santa Cruz Biotechnology), mouse anti-peptidyl-citrulline antibody (MABN328, Merck Millipore, Feltham, UK), and rabbit anti-NF-κB antibody (ab7970, Abcam, 1:100) at 4 °C and then rinsed with PBS.

Techniques: In Vitro, Immunofluorescence, Staining, CCK-8 Assay, Viability Assay, BrdU Incorporation Assay, LDH Cytotoxicity Assay, Cell Cycle Assay, Wound Healing Assay

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Schematic diagram summarizing PAD2-mediated signaling in hCECs.

Techniques:

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 rate of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6, *** p = 0.0002); ( I ) The statistical data of PP interval of WT mice before and after hemorrhagic shock (n = 6, ** p = 0.0053), and the statistical data of PP interval of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6, *** p = 0.0002); ( J ) The statistical data of QT interval of WT mice before and after hemorrhagic shock (n = 6, ** p = 0.009), and the statistical data of PP interval of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6); ( K ) After hemorrhagic shock, Total duration of ventricular tachycardia (left) (n = 6 vs. n = 6, **** p < 0.0001) and Incidence of ventricular fibrillation (right) of WT mice and Pad2 −/− mice.

Journal: The Journal of Trauma and Acute Care Surgery

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

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: 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 rate of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6, *** p = 0.0002); ( I ) The statistical data of PP interval of WT mice before and after hemorrhagic shock (n = 6, ** p = 0.0053), and the statistical data of PP interval of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6, *** p = 0.0002); ( J ) The statistical data of QT interval of WT mice before and after hemorrhagic shock (n = 6, ** p = 0.009), and the statistical data of PP interval of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6); ( K ) After hemorrhagic shock, Total duration of ventricular tachycardia (left) (n = 6 vs. n = 6, **** p < 0.0001) and Incidence of ventricular fibrillation (right) of WT mice and Pad2 −/− mice.

Article Snippet: Lysates were pre-cleared with Protein A/G Agarose Beads (Beyotime, P2012), quantified via BCA, then incubated overnight at 4°C with antibodies: PAD2 (Proteintech, 12110-1-AP), SERCA2a (Abcam, ab3625), or IgG control (Proteintech, 30000-0-AP).

Techniques: Knock-Out, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing, Western Blot

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 hemorrhagic shock model; ( F ) Kaplan-Meier survival rate curve of cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice after hemorrhagic shock, n = 6 for each group, ** p = 0.003; ( G ) Representative electrocardiogram monitoring images of cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice after hemorrhagic shock; ( H ) Before and after hemorrhagic shock, heart beats statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, * p = 0.0363, *** p = 0.0004; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and control mice (right), n = 6 for each group, *** p = 0.0001, *** p = 0.0008; ( I ) Before and after hemorrhagic shock, P amplitude statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, * p = 0.0467, * p = 0.0302; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice (right), n = 6 for each group, **** p < 0.0001, * p = 0.0158; ( J ) Before and after hemorrhagic shock, PP interval statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, *** p = 0.0002; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice (right), n = 6 for each group, **** p < 0.0001, *** p = 0.0009; ( K ) Before and after hemorrhagic shock, QT interval statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, ** p = 0.0077, ** p = 0.0015; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice(right), n = 6 for each group, *** p = 0.0003, *** p = 0.0003.

Journal: The Journal of Trauma and Acute Care Surgery

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

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: 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 hemorrhagic shock model; ( F ) Kaplan-Meier survival rate curve of cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice after hemorrhagic shock, n = 6 for each group, ** p = 0.003; ( G ) Representative electrocardiogram monitoring images of cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice after hemorrhagic shock; ( H ) Before and after hemorrhagic shock, heart beats statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, * p = 0.0363, *** p = 0.0004; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and control mice (right), n = 6 for each group, *** p = 0.0001, *** p = 0.0008; ( I ) Before and after hemorrhagic shock, P amplitude statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, * p = 0.0467, * p = 0.0302; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice (right), n = 6 for each group, **** p < 0.0001, * p = 0.0158; ( J ) Before and after hemorrhagic shock, PP interval statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, *** p = 0.0002; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice (right), n = 6 for each group, **** p < 0.0001, *** p = 0.0009; ( K ) Before and after hemorrhagic shock, QT interval statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, ** p = 0.0077, ** p = 0.0015; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice(right), n = 6 for each group, *** p = 0.0003, *** p = 0.0003.

Techniques: Over Expression, Construct, Injection, Control, Confocal Microscopy, Western Blot, Infection

PAD2 deficiency protects cardiomyocytes from arrhythmia by maintaining Ca 2+ homeostasis. ( A ) Representative diagram of cytosolic Ca 2+ 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 Ca 2+ 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 Ca 2+ 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/F 0 ) over time. ( E ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 , and n = 10 cells from 6 mice in each group. ** p < 0.0001. ( F ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult rat cardiomyocytes infected with Ad-Scramble or Ad-sh-PAD2, ( G ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time, and ( H ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 . n = 6 cells from 6 rats and n = 10 cells from 6 rats, *** p = 0.0003. ( I ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult rat cardiomyocytes infected with Ad-LacZ or Ad-PAD2, ( J ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time, and ( K ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F0. n = 28 cells from 10 rats and n = 25 cells from 10 rats, **** p < 0.0001. ( L ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal of spontaneous calcium waves in adult mouse cardiomyocytes from WT and Pad2 −/− mice. ( M ) Percentage of cells with spontaneous calcium waves. n = 15 cells from 5 mice in each group. ( N ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal of spontaneous calcium sparks in adult mouse cardiomyocytes from WT and Pad2 −/− mice.

Journal: The Journal of Trauma and Acute Care Surgery

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

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: PAD2 deficiency protects cardiomyocytes from arrhythmia by maintaining Ca 2+ homeostasis. ( A ) Representative diagram of cytosolic Ca 2+ 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 Ca 2+ 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 Ca 2+ 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/F 0 ) over time. ( E ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 , and n = 10 cells from 6 mice in each group. ** p < 0.0001. ( F ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult rat cardiomyocytes infected with Ad-Scramble or Ad-sh-PAD2, ( G ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time, and ( H ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 . n = 6 cells from 6 rats and n = 10 cells from 6 rats, *** p = 0.0003. ( I ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult rat cardiomyocytes infected with Ad-LacZ or Ad-PAD2, ( J ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time, and ( K ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F0. n = 28 cells from 10 rats and n = 25 cells from 10 rats, **** p < 0.0001. ( L ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal of spontaneous calcium waves in adult mouse cardiomyocytes from WT and Pad2 −/− mice. ( M ) Percentage of cells with spontaneous calcium waves. n = 15 cells from 5 mice in each group. ( N ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal of spontaneous calcium sparks in adult mouse cardiomyocytes from WT and Pad2 −/− mice.

Techniques: Fluorescence, Infection

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 significant decrease in nuclear Pad2 and a significant increase in cytoplasmic Pad2 after hypoxia exposure (n = 6 per group, **** p < 0.0001). ( D ) Immunofluorescence staining (top) showing the co-localization of Pad2 with PDI in cardiomyocytes with and without hypoxic stimulation. Scale bar = 10 μm (upper) Scale bar = 20 μm (bottom). Pearson’s correlation coefficient was calculated to quantify the degree of co-localization between Pad2 and PDI (bottom). **** p < 0.0001. ( E ) Sarcoplasmic reticulum proteins were separated and extracted, and Pad2 protein levels were quantified using Western blotting. Representative Western blot (top) and average data (bottom) indicate the levels of Pad2 in the sarcoplasmic reticulum. n = 4 independent experiments, * p = 0.014.

Journal: The Journal of Trauma and Acute Care Surgery

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

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: 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 significant decrease in nuclear Pad2 and a significant increase in cytoplasmic Pad2 after hypoxia exposure (n = 6 per group, **** p < 0.0001). ( D ) Immunofluorescence staining (top) showing the co-localization of Pad2 with PDI in cardiomyocytes with and without hypoxic stimulation. Scale bar = 10 μm (upper) Scale bar = 20 μm (bottom). Pearson’s correlation coefficient was calculated to quantify the degree of co-localization between Pad2 and PDI (bottom). **** p < 0.0001. ( E ) Sarcoplasmic reticulum proteins were separated and extracted, and Pad2 protein levels were quantified using Western blotting. Representative Western blot (top) and average data (bottom) indicate the levels of Pad2 in the sarcoplasmic reticulum. n = 4 independent experiments, * p = 0.014.

Techniques: Translocation Assay, Western Blot, Immunofluorescence, Staining, Fluorescence, Isolation

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 neonatal rat cardiomyocytes infected with Ad-sh-Scramble or Ad-sh-PAD2 for 48 hours, followed by normoxia or hypoxia stimulation for 1 hour (n = 6 vs. n = 6 per group, **** p < 0.0001, *** p = 0.0002). ( J ) CoIP (co-immunoprecipitation) analysis showing the interaction of Serca2a protein with a citrullination antibody in neonatal cardiomyocytes under normoxic or hypoxic conditions for 1 hour. ( K ) CoIP (co-immunoprecipitation) analysis showing the interaction of SERCA2a protein with a citrullination antibody in heart tissue under hemorrhagic shock. ( L ) Cardiomyocytes transfected with the pCMV-PAD2-Ca2 mutation plasmid exhibited significantly reduced Pad2 enzyme activity compared to those transfected with the WT pCMV-PAD2 plasmid (n = 5 vs. n = 5 per group, **** p < 0.0001). ( M ) Cardiomyocytes transfected with the pCMV-PAD2-Ca2 mutation plasmid reversed hypoxia-induced reductions in SERCA2a activity compared to those transfected with the WT pCMV-PAD2 plasmid (n = 3 vs. n = 3, ** p = 0.0016, *** p = 0.0005).

Journal: The Journal of Trauma and Acute Care Surgery

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

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: 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 neonatal rat cardiomyocytes infected with Ad-sh-Scramble or Ad-sh-PAD2 for 48 hours, followed by normoxia or hypoxia stimulation for 1 hour (n = 6 vs. n = 6 per group, **** p < 0.0001, *** p = 0.0002). ( J ) CoIP (co-immunoprecipitation) analysis showing the interaction of Serca2a protein with a citrullination antibody in neonatal cardiomyocytes under normoxic or hypoxic conditions for 1 hour. ( K ) CoIP (co-immunoprecipitation) analysis showing the interaction of SERCA2a protein with a citrullination antibody in heart tissue under hemorrhagic shock. ( L ) Cardiomyocytes transfected with the pCMV-PAD2-Ca2 mutation plasmid exhibited significantly reduced Pad2 enzyme activity compared to those transfected with the WT pCMV-PAD2 plasmid (n = 5 vs. n = 5 per group, **** p < 0.0001). ( M ) Cardiomyocytes transfected with the pCMV-PAD2-Ca2 mutation plasmid reversed hypoxia-induced reductions in SERCA2a activity compared to those transfected with the WT pCMV-PAD2 plasmid (n = 3 vs. n = 3, ** p = 0.0016, *** p = 0.0005).

Techniques: Activity Assay, Infection, Immunoprecipitation, Mass Spectrometry, Western Blot, Transfection, Mutagenesis, Plasmid Preparation

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 Ca 2+ 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 electronic pacing; n = 20 from 5 mice in control group, n = 18 from 5 mice in AFM-30a treated group. ( F ) Average Ca 2+ decay Tau of adult mouse cardiomyocytes in response to 10-V, 0.5-Hz (n = 6 vs. n = 6, **** p < 0.0001), 2.0-Hz (n = 6 vs. n = 6, * p = 0.0138), and 3.0 Hz (n = 6 vs. n = 6, ** p = 0.0028) separately. ( G ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult mouse cardiomyocytes treated with 25 μM of AFM-30a or DMSO for 24 hours, ( H ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time. ( I ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 , and n = 10 cells from 6 mice in each group. ** p < 0.0001.

Journal: The Journal of Trauma and Acute Care Surgery

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

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: 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 Ca 2+ 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 electronic pacing; n = 20 from 5 mice in control group, n = 18 from 5 mice in AFM-30a treated group. ( F ) Average Ca 2+ decay Tau of adult mouse cardiomyocytes in response to 10-V, 0.5-Hz (n = 6 vs. n = 6, **** p < 0.0001), 2.0-Hz (n = 6 vs. n = 6, * p = 0.0138), and 3.0 Hz (n = 6 vs. n = 6, ** p = 0.0028) separately. ( G ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult mouse cardiomyocytes treated with 25 μM of AFM-30a or DMSO for 24 hours, ( H ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time. ( I ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 , and n = 10 cells from 6 mice in each group. ** p < 0.0001.

Techniques: Injection, Control, Fluorescence

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