Journal: Journal of the American Heart Association

Article Title: Cardiac Gene Therapy With Phosphodiesterase 2A Limits Remodeling and Arrhythmias in Mouse Models of Heart Failure

doi: 10.1161/jaha.124.037343

Figure Lengend Snippet: Figure 2. Gene therapy with PDE2A prevents cardiac remodeling and dysfunction induced by chronic isoproterenol infusion. A, Schematic representation of the experimental protocol. Mice were injected in the tail vein with 1012 viral particles of a serotype 9 adeno-associated viruses encoding for luciferase or phosphodiesterase 2A3. Fourteen days later, mice injected with AAV9-LUC were implanted subcutaneously with osmotic pumps diffusing either NaCl (NaCl-LUC) or isoproterenol at 60 mg/kg per day (isoproterenol- LUC) and animals injected with AAV9-PDE2A were implanted with pumps delivering isoproterenol at 60 mg/kg per day (isoproterenol- PDE2A). Treatment duration was 14 days (hatched bars). Minipumps were then removed and mice were kept for 2 additional weeks before euthanasia. Cardiac function was evaluated by serial echocardiography before injection of the virus as well as at 14, 28, 35, and 42 days. B, Left panel shows representative blots of PDE2A and vinculin. PDE2A protein expression in heart tissues extracts measured by Western blot and the bar graph represents the ratios of PDE2A over vinculin quantified expressed as mean±SEM in NaCl-LUC, isoproterenol-LUC and isoproterenol-PDE2A groups. C, Heart weight and lung weight over tibia length ratio expressed as mean±SEM. D, Time course of the normalized ratio of calculated left ventricular weight over body weight and normalized ejection fraction in NaCl-LUC, isoproterenol-LUC and isoproterenol-PDE2A mice. Number of mice is indicated in the brackets. E, Panel on the left, representative images of Masson’s trichrome staining (scale bar 100 μm), bar graph on the left represents quantifications of interstitial fibrosis as mean±SEM in NaCl-LUC (n=9), isoproterenol-LUC (n=9), and isoproterenol+PDE2A (n=9) groups. Statistical significance is indicated by the P value (* vs NaCl-LUC and † vs isoproterenol-LUC) determined using Welch’s t test (B), Kruskal– Wallis followed by a Dunn’s test (C panel bottom and E), 2-way ANOVA followed by a Tukey’s test (D), 1-way ANOVA followed by a Tukey’s test (C panel top). AAV9 indicates serotype 9 adeno-associated viruses; BW, body weight; HW, heart weight; Iso-LUC, isoproterenol-luciferase; Iso-PDE2A, isoproterenol-phosphodiesterase 2A; LUC, luciferase; LVW, left ventricular weight; LW, lung weight; PDE2A, phosphodiesterase 2A; and TL, tibia length.

Article Snippet: The primary antibodies used were as follows: (1) Figure 1: rabbit anti- PDE2A, Fabgennix; PDE2A- 101AP diluted at 1/1000; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; (2) Figure 2: goat antiPDE2A, Santa Cruz, SC- 17228 diluted at 1/1000; mouse anti- vinculin, V9131 Sigma- Aldrich diluted at 1/1000; (3) Figure 3: rabbit anti- PDE2A, Fabgennix, PDE2A- 101AP diluted at 1/1000; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; (4) Figure S1: rabbit anti- flag L5, Novus Biologicals, NBP1- 06712 diluted at 1/500; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; rabbit anti- PDE2A, Proteintech, 55 306- 1- AP diluted at 1/1000; rabbit anti- flag L5, Novus Biologicals, NBP1- 06712 diluted at 1/500; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; (5) Figure S2B: rabbit anti- PDE2A, Proteintech, 55 306- 1- AP diluted at 1/1000; (6) Figure S3: rabbit antiPDE2A, Proteintech, 55 306- 1- AP diluted at 1/1000; and (7) Figure S4: rabbit anti- PDE4A AC55 (gift from Dr. Marco Conti, University of California San Francisco, San Francisco, CA) diluted at 1/1000; rabbit anti- PDE4B (gift from Dr. Marco Conti) diluted at 1/10000; mouse antiPDE4D (gift from Dr Marco Conti) diluted at 1/1000 and rabbit anti- PDE3A (gift from Dr Chen Yan, University of Rochester, Rochester, NY) diluted at 1/10000.

Techniques: Injection, Luciferase, Virus, Expressing, Western Blot, Staining

Journal: Journal of the American Heart Association

Article Title: Cardiac Gene Therapy With Phosphodiesterase 2A Limits Remodeling and Arrhythmias in Mouse Models of Heart Failure

doi: 10.1161/jaha.124.037343

Figure Lengend Snippet: Figure 3. PDE2A overexpression limits maladaptive remodeling and cardiac dysfunction induced by chronic isoproterenol and phenylephrine infusion. A, Schematic representation of the experimental protocol. Mice were injected with 1012 viral particles of serotype 9 adeno-associated viruses encoding for Luciferase or phosphodiesterase 2A3. Fourteen days later, mice were implanted with osmotic minipumps diffusing either NaCl (NaCl-LUC) or isoproterenol+phenylephrine at 30 mg/kg per day each (isoproterenol+phenylephrine-LUC and isoproterenol + phenylephrine-PDE2A). Minipumps were removed 2 weeks later and mice were euthanized. Cardiac function was assessed throughout the protocol using echocardiography before the injection of the virus and at 2, 3, 4, and 6 weeks. B, Representative blots of PDE2A and GAPDH on the left panel are shown and the bar graph represents ratios of PDE2A over GAPDH quantifications expressed as mean±SEM in a subset of the animals treated with NaCl-LUC, isoproterenol+phenylephrine-LUC, and isoproterenol+phenylephrine-PDE2A. C, Heart weight and lung weight over tibia length ratios expressed as mean±SEM. D, Time course of the normalized ratios of calculated left ventricular weight (over body weight and normalized ejection fraction in NaCl-LUC, isoproterenol+phenylephrine-LUC, and isoproterenol+phenylephrine-PDE2A mice). Number of mice is indicated in the brackets. E, Top panel shows representative images of Masson’s trichrome staining (scale bar 200 μm), the middle panel depicts representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (scale bar 100 μm) detecting apoptotic nuclei in green costained with the glycocalix marker wheat germ agglutinin in red and nuclei in blue colored with Hoechst. Bar graphs representing mean±SEM quantifications of interstitial fibrosis and apoptotic nuclei in NaCl-LUC (10), isoproterenol+phenylephrine- LUC (9) and isoproterenol+phenylephrine-PDE2A (9) mice are depicted. Statistical significance indicated by the P value (* vs NaCl- LUC and † vs isoproterenol+phenylephrine-LUC) was determined using Welch’s t test (B), Kruskal–Wallis followed by a Dunn’s test (C), 2-way ANOVA followed by a Tukey’s test (D), Welch’s 1-way ANOVA followed by a Dunnett’s test (E). AAV9 indicates serotype 9 adeno-associated viruses; BW, body weight; HW, heart weight; Iso-Phe-LUC, isoproterenol-phenylephrine-luciferase; Iso-Phe- PDE2A, isoproterenol-phenylephrine-phosphodiesterase 2A; LUC, luciferase; LVW, left ventricular weight; LW, lung weight; ns, not significant; PDE2A, phosphodiesterase 2A; and TL, tibia length.

Article Snippet: The primary antibodies used were as follows: (1) Figure 1: rabbit anti- PDE2A, Fabgennix; PDE2A- 101AP diluted at 1/1000; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; (2) Figure 2: goat antiPDE2A, Santa Cruz, SC- 17228 diluted at 1/1000; mouse anti- vinculin, V9131 Sigma- Aldrich diluted at 1/1000; (3) Figure 3: rabbit anti- PDE2A, Fabgennix, PDE2A- 101AP diluted at 1/1000; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; (4) Figure S1: rabbit anti- flag L5, Novus Biologicals, NBP1- 06712 diluted at 1/500; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; rabbit anti- PDE2A, Proteintech, 55 306- 1- AP diluted at 1/1000; rabbit anti- flag L5, Novus Biologicals, NBP1- 06712 diluted at 1/500; rabbit anti- GAPDH, Cell Signaling, 2118 diluted at 1/1000; (5) Figure S2B: rabbit anti- PDE2A, Proteintech, 55 306- 1- AP diluted at 1/1000; (6) Figure S3: rabbit antiPDE2A, Proteintech, 55 306- 1- AP diluted at 1/1000; and (7) Figure S4: rabbit anti- PDE4A AC55 (gift from Dr. Marco Conti, University of California San Francisco, San Francisco, CA) diluted at 1/1000; rabbit anti- PDE4B (gift from Dr. Marco Conti) diluted at 1/10000; mouse antiPDE4D (gift from Dr Marco Conti) diluted at 1/1000 and rabbit anti- PDE3A (gift from Dr Chen Yan, University of Rochester, Rochester, NY) diluted at 1/10000.

Techniques: Over Expression, Injection, Luciferase, Virus, Staining, TUNEL Assay, Marker

Journal: Cell reports

Article Title: Beta-Catenin Causes Adrenal Hyperplasia by Blocking Zonal Transdifferentiation

doi: 10.1016/j.celrep.2020.107524

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunostaining was done using the following primary antibodies: anti-β-catenin (mouse monoclonal, BD Biosciences, 610153; and rabbit polyclonal, Abcam, ab16051), anti-GFP (chicken polyclonal, Aves Labs, GFP-1020), anti-activated Caspase 3 (rabbit monoclonal, BD Biosciences, 559565), anti-Dab2 (mouse monoclonal, BD Biosciences, 610464), anti-Akr1b7 (rabbit polyclonal, kindly provided by Dr. Pierre Val and Dr. Antoine Martinez ( ; )), anti-Gαq (rabbit polyclonal, Abcam, ab75825), anti-AS (rabbit polyclonal, kindly provided by Dr. Celso E. Gomez-Sanchez ( )), anti-Ki67 (rabbit monoclonal, Lab Vision, RM-9106), anti-Lef1 (rabbit monoclonal, Abcam, ab137872), anti-Phospho-(Ser/Thr) PKA Substrate Antibody (rabbit polyclonal, Cell Signaling Technology, 9621), anti-β-catenin(ex3) (rabbit monoclonal, Abcam, ab32572), anti-PDE2A (rabbit polyclonal, Proteintech, 55306–1-AP).

Techniques: Recombinant, Reverse Transcription, TUNEL Assay, In Situ, SYBR Green Assay, Software

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

Article Title: Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure

doi: 10.1073/pnas.1800996115

Figure Lengend Snippet: PDE2 inhibition augments cardiac cGMP, but not cAMP, levels in vivo and promotes the antihypertrophic effect of NO, but not ANP, in isolated cardiomyocytes. ( A ) Cardiomyocyte area in cells isolated from neonatal WT mice treated with Ang II (100 nM) for 24 h in the absence and presence of the NO donor DETA (10 μM), ANP (1 μM), or BNP (1 μM) under basal conditions or following treatment with BAY 60-7550 (100 nM). ( B ) Cyclic nucleotide levels in whole-heart homogenates from sham mice or animals subjected to AAC for 6 wk (6w) in the absence and presence of BAY 60-7550 (10 mg⋅kg −1 ⋅d −1 p.o., initiated at 3 wk). ( C ) Immunoblot analysis of PDE2A expression in whole-heart homogenates from mice subjected to AAC for 6w in the absence and presence of BAY 60-7550 (10 mg⋅kg −1 ⋅d −1 p.o., initiated at 3 wk). Data are expressed as mean ± SEM with analysis by one-way ANOVA with a Bonferroni post hoc test. * P < 0.05 versus Ang II + DETA ( A ); ** P < 0.01 versus sham versus ACC, # P < 0.05 versus AAC ( B ); * P < 0.05 versus sham ( C ) ( n = 6).

Article Snippet: PDE2A protein expression was determined by immunoblot using primary anti-PDE2A antibody (1:500; Santa Cruz Biotechnology) and secondary horseradish peroxidase-conjugated anti-goat IgG antibody (1:10,000; Santa Cruz Biotechnology).

Techniques: Inhibition, In Vivo, Isolation, Western Blot, Expressing

Journal: Science Advances

Article Title: Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics

doi: 10.1126/sciadv.add5799

Figure Lengend Snippet: ( A ) Total cAMP activity from RV basal (blue) and apical (orange) regions in male and female hearts. N = 5 hearts in each group, * P < 0.05, by two-way ANOVA with multiple pairwise comparisons. Representative Western blots ( B ) and normalized mean data ( C and D ) for PDE3A, PDE4A, PDE4B, and PDE4D normalized to tubulin expression, in male and female mouse whole ventricles (C) and from RV basal and apical regions (D). N = 5 to 6 hearts in each group. * P < 0.05 and ** P < 0.01, by one-way ANOVA.

Article Snippet: The following primary antibodies were used for immunoblotting: PDE3A (112AP, FabGennix International Inc., Frisco, TX), PDE4A and PDE4B (gifts from M. Conti, University of California at San Francisco), PDE4D (ab14613, Abcam, Cambridge, MA), and γ-tubulin (T6557, Sigma-Aldrich, St. Louis, MO).

Techniques: Activity Assay, Western Blot, Expressing

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Investigation of an 18 F-labelled Imidazopyridotriazine for Molecular Imaging of Cyclic Nucleotide Phosphodiesterase 2A

doi: 10.3390/molecules23030556

Figure Lengend Snippet: IC 50 values of the novel 2-fluoroethoxy derivative TA5 for the inhibition of human PDE2A and human PDE10A compared to our already published data for the lead compound TA1 and the PDE2A ligands TA2 – 4 [ 13 , 19 , 20 ].

Article Snippet: The inhibitory potencies of TA1 – 5 for human recombinant PDE2A and PDE10A proteins were determined by BioCrea GmbH (Radebeul, Germany) [ ].

Techniques: Inhibition