Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphodiesterase 5 Associates With β2 Adrenergic Receptor to Modulate Cardiac Function in Type 2 Diabetic Hearts

doi: 10.1161/JAHA.119.012273

Figure Lengend Snippet: Sildenafil treatment improves E‐C coupling protein activation and expression in mice fed a high fat diet (HFD). Images and quantification of Western blots for phosphodiesterase 5A ( PDE 5A), phosphodiesterase 3A ( PDE 3A), phospho‐serine 282 (p282) and total cardiac myosin binding protein C ( cMyBP ‐C), phospho‐serine 23/24 (p23/24) and total troponin I (TnI), sarcoplasmic‐endoplasmic reticulum calcium ATP ase 2A ( SERCA 2A), and phospho‐serine 16 (p16), and total phospholamban ( PLB ) for normal chow (NC) and HFD mice ( A and B ) followed by that for HFD and HFD ‐sildenafil mice ( C and D ). Total protein normalized to glyceraldehyde 3‐phosphate ( GAPDH ). * P ≤0.05, ** P ≤0.01, *** P ≤0.001 by unpaired t test. n=3 mice per group.

Article Snippet: Primary antibodies instituted included PDE5A (2395, Cell Signaling) with β2AR (SC‐271322, Santa Cruz) for PDE5A:β2AR PLA, phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) with β2AR (SC‐570, Santa Cruz) for PDE3A:β2AR PLA, and PDE5A (2395, Cell Signaling) with phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) for PDE5:PDE3 PLA.

Techniques: Activation Assay, Expressing, Western Blot, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphodiesterase 5 Associates With β2 Adrenergic Receptor to Modulate Cardiac Function in Type 2 Diabetic Hearts

doi: 10.1161/JAHA.119.012273

Figure Lengend Snippet: Phosphodiesterase 5 preferentially interacts with β2 adrenergic receptor (β2 AR) and its signaling cascade. A through D , Average maximal fluorescence resonant energy transfer (FRET) response of the cGMP probe to isoproterenol, cG i500 in isolated neonatal cardiomyocytes from wild‐type ( WT) , β1 adrenergic receptor (β1AR) knockout (β1 KO) , β2AR knockout (β2 KO) , and β3 adrenergic receptor knockout (β3 KO) mice. n≥30 cells per group. * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001 by 1‐way ANOVA with Tukey post‐test. E , Average maximal FRET response of the cGMP probe to isoproterenol, cG i500 in isolated NC and HFD ACM s with pretreatment of the β1 AR inhibitor, CGP ‐20712 ( CGP ), or the β2 AR inhibitor, ICI ‐118551 ( ICI ). n≥20 cells per group. †††† P ≤0.0001 compared with all other groups, * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001 by 1‐way ANOVA with Tukey post‐test. F , Ejection fraction and ( G ) fractional shortening echocardiogram measurements of WT and β2 KO mice fed normal chow ( NC) or high fat diet ( HFD) after 4.5 months of special diet. H , Heart mass/tibia length ratio of WT and β2 KO mice fed NC or HFD for 4.5 months. n≥7 mice per group. * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001 by 1‐way ANOVA with Tukey post‐test. I , Interstitial cardiac fibrosis percentage in WT and β2 KO mice fed NC or HFD for 4.5 months. n=3 mice per group. Kruskal–Wallis test with Dunn's post‐test performed. J , Ejection fraction and ( K ) fractional shortening echocardiogram measurements of WT and β2 KO mice fed NC and HFD for 4.5 months after isoproterenol injection followed by sildenafil injection. n≥8 mice per group. * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001 by 1‐way ANOVA with Tukey post‐test.

Article Snippet: Primary antibodies instituted included PDE5A (2395, Cell Signaling) with β2AR (SC‐271322, Santa Cruz) for PDE5A:β2AR PLA, phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) with β2AR (SC‐570, Santa Cruz) for PDE3A:β2AR PLA, and PDE5A (2395, Cell Signaling) with phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) for PDE5:PDE3 PLA.

Techniques: Fluorescence, Isolation, Knock-Out, Injection

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphodiesterase 5 Associates With β2 Adrenergic Receptor to Modulate Cardiac Function in Type 2 Diabetic Hearts

doi: 10.1161/JAHA.119.012273

Figure Lengend Snippet: Phosphodiesterase 3 (PDE3) displays an increased association with the phosphodiesterase 5 (PDE5)–β2 adrenergic receptor (β2 AR) protein complex in high fat diet (HFD) cardiomyocytes. A , Representative images and ( B ) quantification of coimmunoprecipitation of Flag‐tagged βAR s from rabbit adult ventriculomyocytes (AVM s) expressing Flag‐mβ2 AR or CFP (cyan fluorescent protein) – and Flag‐tagged mouse β1 adrenergic receptor (β1AR), with detection of PDE 5A. n=3 rabbits. * P ≤0.05 by unpaired t test. Proximity ligation assay ( PLA ) representative images and quantification in normal chow (NC) and HFD AVM s for the interaction between PDE 5A and β2 AR ( C ), between PDE 3A and β2 AR ( D ), and between PDE 3A and PDE 5A ( E ). Scale bar=10 μm. Red= PLA signal. Blue= DAPI . n=18 cells per group. **** P ≤0.0001 by unpaired t test.

Article Snippet: Primary antibodies instituted included PDE5A (2395, Cell Signaling) with β2AR (SC‐271322, Santa Cruz) for PDE5A:β2AR PLA, phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) with β2AR (SC‐570, Santa Cruz) for PDE3A:β2AR PLA, and PDE5A (2395, Cell Signaling) with phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) for PDE5:PDE3 PLA.

Techniques: Expressing, Proximity Ligation Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphodiesterase 5 Associates With β2 Adrenergic Receptor to Modulate Cardiac Function in Type 2 Diabetic Hearts

doi: 10.1161/JAHA.119.012273

Figure Lengend Snippet: Inhibition of phosphodiesterase 5 ( PDE 5) by sildenafil improves contractility in a β2 adrenergic receptor (β 2 AR)– dependent mechanism ( A ) depiction of the pathway sildenafil uses in mice fed a high fat diet to improve function. CaMKII indicates calcium/calmodulin kinase II; G i , inhibitory G protein; G s , stimulatory G protein; PKA, protein kinase A; PKG, protein kinase G; PDE3, phosphodiesterase 3; β 1 AR, β1 adrenergic receptor.

Article Snippet: Primary antibodies instituted included PDE5A (2395, Cell Signaling) with β2AR (SC‐271322, Santa Cruz) for PDE5A:β2AR PLA, phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) with β2AR (SC‐570, Santa Cruz) for PDE3A:β2AR PLA, and PDE5A (2395, Cell Signaling) with phosphodiesterase 3A (NIH, PDE3A‐CT aa1098‐1115) for PDE5:PDE3 PLA.

Techniques: Inhibition

Journal: bioRxiv

Article Title: β2-Adrenergic Biased Agonist Nebivolol Inhibits the Development of Th17 and the Response of Memory Th17 Cells in an NF-κB-Dependent Manner

doi: 10.1101/2024.09.08.611829

Figure Lengend Snippet: Memory Th cells were not activated or activated with antiCD3/antiCD28/antiCD2 for five days A) Expression of ADRB1 -3 in RNA extracted from enrichment memory Th cells shown as the relative amounts normalized to housekeeping RNA. Pooled data are expressed as the Mean ± SEM of ten independent biological experiments. B) Expression of ADRB2 at mRNA levels from Memory Th cells with or without treatment after five days of culture, shown as the relative amounts normalized to housekeeping RNA and compared to the Act group. The data of this figure is representative of three experiments. C) A representative western blot data of equal amounts of protein from the cell lysates was shown for β2AR and GAPDH as a loading control. D) A representative band intensity of five biological experiments was quantified and shown corrected to the loading control as a ratio. The bars show the Mean ± SEM. ANOVA followed by Tukey’s multiple comparisons tests.

Article Snippet: Antibodies were against human Phospho-ser529 NF-κB p65 (1/500, clone A21012B Biolegend), NF-κB p65 Antibody (1/1000, clone 14G10A21, Biolegend), phospho-Ser133 CREB1 (1/700, rabbit polyclonal, Cusabio, Cedarlane), phospho-Ser412-β-arrestin1 (1/1000, clone mAb #2416 Cell Signaling Tech., Danvers, USA), β2AR (1/500, clone 4A6C9, Novusbio, Cedarlane), GAPDH (1/15000, 10B4E3, MA000071M1m, Cusabio, Cedarlane) and α□tubulin (1/1000, Santa Cruz Biotechnology Inc., USA).

Techniques: Expressing, Western Blot, Control