Journal: iScience

Article Title: Atrial natriuretic peptide signaling co-regulates lipid metabolism and ventricular conduction system gene expression in the embryonic heart

doi: 10.1016/j.isci.2023.108748

Figure Lengend Snippet: ANP increases VCS marker expression and cell proliferation in E11.5 ventricular cell cultures (A) Representative western blots for Cx40, HCN4 and GAPDH levels in E11.5 ventricular cell cultures treated with or without ANP and or NPRA inhibitor (A71915 or A7) for 3 days. (B and C) Quantification of Cx40 and HCN4 expression levels normalized to that of GAPDH. Levels in treated groups are presented as fold changes compared to the control group. Bars represent mean ± SEM, n = 3, ∗p < 0.05, One-way ANOVA, Dunnett’s multiple comparisons test. (D and E) In-cell western analysis of Cx40 and sarc. myosin levels in E11.5 ventricular cell cultures treated with or without ANP and NPRA inhibitor (A7) for 1 day. Bars represent mean ± SEM, n = 3–4, ∗p < 0.05, ∗∗p < 0.01, One-way ANOVA, Dunnett’s multiple comparisons test. (F) Click-IT EdU nuclear labeling of cultures treated with or without ANP and A7. VCS cardiomyocytes (Cx40+Sarc. MHC+) were identified by co-staining with Cx40 and sarc. MHC antibodies. Arrow indicates a Cx40+ VCS cardiomyocyte negative for Edu labeling and arrowhead indicates an Edu labeled VCS cardiomyocyte. Note the Cx40 localization in the plasma membrane of two adjacent non-dividing cells.Scale bars = 20μm. (G) Examples of VCS cells undergoing cell division in cultures treated with ANP. Mitotic figures and cell division were visualized by pan-phospho-aurora kinase (AURK) immunostaining and Hoechst nuclear staining. VCS were cells identified using antibodies for sarcomeric MHC and Cx40. Note the cytoplasmic localization of Cx40 in VCS cells undergoing mitosis and cytokinesis. Scale bars = 20 μm. (H–J) Percentage distribution of EdU labeled (H), AURK+ (I) and total (J) VCS cardiomyocytes in E11.5 ventricular cell culture treated with or without ANP and A7 for 1 day. Bars represent mean ± SEM, n = 3, ∗p < 0.05, ∗∗p < 0.01, One-way ANOVA, Dunnett’s multiple comparisons test.

Article Snippet: NPRA antagonist A71915 (Bachem, Cat#H-3048) stock solution was made by dissolving 0.5mg of the compound in 0.5mL of sterile water.

Techniques: Marker, Expressing, Western Blot, Control, In-Cell ELISA, Labeling, Staining, Clinical Proteomics, Membrane, Immunostaining, Cell Culture

Journal: iScience

Article Title: Atrial natriuretic peptide signaling co-regulates lipid metabolism and ventricular conduction system gene expression in the embryonic heart

doi: 10.1016/j.isci.2023.108748

Figure Lengend Snippet: Effects of ANP on metabolic events in embryonic ventricular cells (A) JC-10 staining of an unfixed E11.5 ventricular cross section to monitor the mitochondrial membrane potential in compact (CM) and trabecular (TM) regions. Scale bar = 50μm. (B) Quantification of JC-10 (590nm) fluorescence intensities normalized to Hoechst fluorescence in TM and CM. Bars represent mean ± SEM, n = 3, ∗∗∗p < 0.0005, Student’s unpaired t test. (C and D) Representative traces of extracellular acidification rates (ECAR, C) and oxygen consumption rates (OCR, D). E11.5 ventricular cell cultures treated with or without ANP and A71915 in 10%FBS-DMEM for 24 h. After this period, the medium was replaced with substrate limited DMEM supplemented with 10mM glucose, 1mM pyruvate and 2mM glutamine for 1 h prior to performing seahorse assays as described in . (E and F) Analysis of ECAR to assess the changes in glycolysis (E) and glycolytic capacity (F) in E11.5 ventricular cell cultures treated with or without ANP and A71915. ECAR values were normalized to the total protein. Bars represent mean ± SEM, n = 3, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001. One-way ANOVA, Tukey’s multiple comparisons tests. (G and H) Analysis of OCR in cell culture to demonstrate changes in basal (G) and maximal (H) mitochondrial oxidative phosphorylation by ANP. OCR values were normalized to the total protein. Bars represent mean ± SEM, n = 3, ∗∗∗∗p < 0.0001. One-way ANOVA, Tukey’s multiple comparisons tests. (I) OCR/ECAR ratio to demonstrate the shift of metabolism from a relatively quiescent stage to a higher energy metabolic state by ANP in embryonic ventricular cells. (J and K) Analysis of fatty acid oxidation (FAO) in E11.5 ventricular cell cultures treated with or without ANP and A71915. seahorse assay shows increased OCR in ANP-treated cell culture. Bars represent mean ± SEM, n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. One-way ANOVA, Tukey’s multiple comparisons tests.

Article Snippet: NPRA antagonist A71915 (Bachem, Cat#H-3048) stock solution was made by dissolving 0.5mg of the compound in 0.5mL of sterile water.

Techniques: Staining, Membrane, Fluorescence, Cell Culture, Phospho-proteomics

Journal: iScience

Article Title: Atrial natriuretic peptide signaling co-regulates lipid metabolism and ventricular conduction system gene expression in the embryonic heart

doi: 10.1016/j.isci.2023.108748

Figure Lengend Snippet:

Article Snippet: NPRA antagonist A71915 (Bachem, Cat#H-3048) stock solution was made by dissolving 0.5mg of the compound in 0.5mL of sterile water.

Techniques: Diagnostic Assay, Recombinant, Modification, Membrane, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 1. Comparison of NPRA and NPRC protein expression. (A) Protein expression of NPRA and NPRC were visualized by western blotting in membrane enriched fractions prepared from a variety of mouse tissues: gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), heart, liver, kidney, and lung. (B) BAT membrane enriched fractions from wildtype and NPRC−/− mice fed either standard chow or HFD were used to visualize NPRA and NPRC protein levels. Šídák’s multiple comparisons test for two-way ANOVA indicated on graph. * P < 0.05, *** P < 0.001, **** P < 0.0001. (C) BAT protein from chow- and HFD-fed mice was separated into membrane and cytosolic enriched fractions. NPRA and NPRC were assessed by western blotting. (D) HIB-1B cells were transfected with the indicated plasmids and treated with adipocyte differentiation cocktail for 48 h. Cells were then treated with the 2.5 µM MG-132 for 16 h. Flag-NPRA and NPRC were assessed from whole-cell lysates by western blotting. Šídák’s multiple comparisons test for one-way ANOVA indicated on graph. ** P < 0.01, *** P < 0.001.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Comparison, Expressing, Western Blot, Membrane, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 2. NPRC immunoprecipitates with NPRA and NPRB. HEK 293FT cells were transfected with plasmids expressing Flag-tagged natriuretic peptide receptors. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag Affinity agarose. (A) Endogenous NPRC coimmunoprecipitated with hNPRA-Flag, hNPRB-Flag, or VEGFR1-Flag. (B) Endogeneous NPRC coimmunoprecipitated with GC-C-Flag. (C) NPRC band intensity to input of NPRC was calculated through Image J software was presented in the right panel. Šídák’s multiple comparisons test for one-way ANOVA comparing all samples to the negative control VEGFR1 for western blots in A and B are indicated on graph. *P < 0.05, ** P < 0.01, *** P < 0.001. (D) hNPRA-Flag expressing HEK 293FT cells were treated with ANP (ANP1-28, 200 nM) or a truncated form of ANP that preferentially binds NPRC (ANP4-23, 200 nM) for 30 min. Neither ANP1-28 or ANP4-23 affected the ability of endogenous NPRC to coimmunoprecipitate with hNPRA-Flag. (E) Endogenous NPRC coimmunoprecipitated with Flag-rNPRA and a truncated version of Flag- rNPRA lacking the c-terminal amino acids 528-1057 [Flag-rNPRA(1-527)].

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Transfection, Expressing, Immunoprecipitation, Software, Negative Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 3. Endogenous natriuretic peptide receptor expression in cell models used. Expression of NPRA, NPRB, and NPRC were detected by western blot in HeLa, HEK293FT, and HAP-1 cells. β-actin was used as the loading control.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Expressing, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 6. Coexpression of NPRC reduces ANP-evoked cGMP production in plasma membranes. Plasma membranes were made from HEK-293T cells transfected with either NPRA-flag plus pcDNA3-Clover control or NPRA-flag plus NPRC-flag. Plasma membranes were made without or with (SI Appendix, Fig. S3) phosphatase inhibitors. Co-expression of hNPRC along with hNPRA reduced the cGMP generated in the presence of 100 nM ANP (P = 0.0163, test for difference in slopes between NPRA and NPRA+NPRC; Šídák’s multiple comparisons test comparing NPRA vs. NPRA+NPRC are indicated on the graphs, ** P < 0.01.). Untransfected cells did not appear to generate cGMP. Western blot of membranes used in the ANP-stimulated cGMP assay.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Clinical Proteomics, Transfection, Control, Expressing, Generated, Western Blot

Journal: Redox biology

Article Title: BNP protects against diabetic cardiomyopathy by promoting Opa1-mediated mitochondrial fusion via activating the PKG-STAT3 pathway.

doi: 10.1016/j.redox.2023.102702

Figure Lengend Snippet: Fig. 11. Schematic figure showing that there is an increased level of BNP at the early stage of diabetic cardiomyopathy as a compensatory protection. BNP binds to its receptor NPRA and activates PKG that consequently interacts with STAT3, which phosphor ylates and activates STAT3-Opa1 signaling and then enhances mitochondrial fusion. Subsequently, BNP- induced mitochondrial fusion inhibits mitochondrial dysfunction and mitigates diabetic cardiomyopathy. Mito, mitochondrial; ROS, reactive oxygen species; △Ψm, mitochondrial membrane potential.

Article Snippet: SiRNAs against BNP (#sc-62023, Santa Cruz Biotechnology), Opa1 (sense: CCAGCAAGGUUAGCUGCAATT; antisense: UUGCAGCUAACCUUGCUGGTT), STAT3 (#sc-270,027, Santa Cruz Biotechnology), PKG (sense: CGAAGAUUCUCAUGCUCAA; antisense: UUGAGCAUGAGAAUCUUCG), NPRA (#sc-40126, Santa Cruz Biotechnology) and negative control siRNA were transfected into primary cardiomyocytes by using Lipofectamine RNAiMAX reagent (Invitrogen).

Techniques: Membrane

Journal: Redox biology

Article Title: BNP protects against diabetic cardiomyopathy by promoting Opa1-mediated mitochondrial fusion via activating the PKG-STAT3 pathway.

doi: 10.1016/j.redox.2023.102702

Figure Lengend Snippet: Fig. 10. PKG was essential for BNP-induced activation of STAT3-Opa1 signaling pathway in HG-treated cardiomyocytes. (A–B) Representative blot images and quantitative analysis of PKG. (C–D) PKG activity. (E–F) Representative blot images and quantitative analysis of PKG, NPRA, p-STAT3, STAT3, and Opa1. (G) Confocal images of mitochondrial morphology stained with MitoTracker Green (upper) and mitochondrial ROS stained with MitoSOX Red (below). (H) Averaged mito chondrial number per cell. (I) Mean size of mitochondria (fold over HG-Con RNAi). (J) Quantitative analysis of MitoSOX Red fluorescence density (fold over HG-Con RNAi). (K) An Annexin V-FITC/PI labeling was used to determine cellular apoptosis by flow cytometry. (L) Quantification of apoptotic cells. (M − N) Co-IP revealed PKG-STAT3 interaction. Results are expressed as mean ± SEM. n = 3–8 per group. **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNAs against BNP (#sc-62023, Santa Cruz Biotechnology), Opa1 (sense: CCAGCAAGGUUAGCUGCAATT; antisense: UUGCAGCUAACCUUGCUGGTT), STAT3 (#sc-270,027, Santa Cruz Biotechnology), PKG (sense: CGAAGAUUCUCAUGCUCAA; antisense: UUGAGCAUGAGAAUCUUCG), NPRA (#sc-40126, Santa Cruz Biotechnology) and negative control siRNA were transfected into primary cardiomyocytes by using Lipofectamine RNAiMAX reagent (Invitrogen).

Techniques: Activation Assay, Activity Assay, Staining, Fluorescence, Labeling, Flow Cytometry, Co-Immunoprecipitation Assay