Grisanti et al. 2014 Cardiac Transcript Data

This file contains whole transcriptome data generated from the hearts of C57BL6/J mice that received infusion of the beta adrenergic receptor agonist isoproterenol (Iso; 3mg/kg/day) for 1 or 2 weeks. Sheet 1 contains the 1 week Iso versus vehicle data and sheet 2 contains the 2 week Iso versus vehicle data

Effect of (A) TNFα and (B) LTα gene deletion upon left ventricular ejection fraction (LVEF) determined 3 or 7 days after MI.

<p><b>N = 14–16/group. </b>*P<0.05 vs. WT at the same time point.</p

Time course of post-MI (A) TNFα and (B) LTα production.

<p>N = 14–16/group. *P<0.05, **P<0.01 vs. control.</p

Effect of TNFR gene deletion upon cardiac function 3 days following MI.

<p>Genetic deletion of TNFR1, but not TNFR2, significantly attenuated post-MI (at day 3) cardiac dysfunction, as determined by left ventricular ejection fraction (LVEF, B) and fractional shortening (FS, B). N = 15–16/group. **P<0.01 vs. own sham MI control; ^$P<0.05, ^$$P<0.01 vs. WT at the same time point.</p

Cardiac GRK2 Expression in Knockout Mice.

<p>(<b>A</b>) Whole heart GRK2 expression from MerCreMer (MCM), GRK2fl/fl (GRK2f/f) and GRK2iKO (inducible KO) groups before and after the treatment with tamoxifen. * <i>p</i><0.05 vs. GRK2f/f, MCM and GRK2iKO treated with vehicle groups. (<b>B</b>) GRK2 expression in cardiomyocytes isolated from GRK2iKO and GRK2f/f control mice. ** <i>p</i><0.05 vs. GRK2f/f. (<b>C</b>) Whole heart GRK2 expression from constitutive GRK2KO mice and GRK2fl/fl control mice. ** <i>p</i><0.01 vs. GRK2f/f.</p

Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine

In heart failure, the β-adrenergic receptors (βARs) become desensitized and uncoupled from heterotrimeric G proteins. This process is initiated by G protein-coupled receptor kinases (GRKs), some of which are upregulated in the failing heart, making them desirable therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine, was previously identified as a GRK2 inhibitor. Utilizing a structure-based drug design approach, we modified paroxetine to generate a small compound library. Included in this series is a highly potent and selective GRK2 inhibitor, <b>14as</b>, with an IC₅₀ of 30 nM against GRK2 and greater than 230-fold selectivity over other GRKs and kinases. Furthermore, <b>14as</b> showed a 100-fold improvement in cardiomyocyte contractility assays over paroxetine and a plasma concentration higher than its IC₅₀ for over 7 h. Three of these inhibitors, including <b>14as</b>, were additionally crystallized in complex with GRK2 to give insights into the structural determinants of potency and selectivity of these inhibitors

GRK5W30A demonstrates altered nuclear translocation <i>in vivo</i>.

<p>(<b>A</b>) Total cell lysates from GRK5KO injected with Ad-GRK5W30A into their LV free wall taken 10 days post-injection. (<b>B</b>) Nuclear lysates from mice with cardiac expression of only GRK5W30A that had received 72 hr of chronic PBS or AngII infusion were immunoblotted for GRK5. (<b>C</b>) Quantitative analysis of the nuclear lysates for nuclear GRK5 accumulation normalized to fibrillarin and reported as fold change. n = 8. (<b>D</b>) HW/BW ratio following 3 days of continuous PBS or AngII infusion for mice expressing GRK5W30A. (<b>E</b>) Total cell lysates from GRK5KO mice injected with Ad-GRK5 CTPB into their LV free wall taken 10 days post-injection. (<b>F</b>) Nuclear lysates from mice cardiac specific expression of only GRK5 CTPB that had received 72 hr of chronic PBS or AngII infusion were immunoblotted for GRK5. (<b>G</b>) Quantitative analysis of the nuclear lysates for nuclear GRK5 accumulation normalized to fibrillarin and reported as fold change. *p<0.05, student’s t test, n = 6 (H) HW/BW ratio following 3 days of continuous PBS or AngII infusion for mice expressing GRK5CTPB. *p<0.05, student’s t test, n = 6.</p

Effect of TNFR gene deletion upon infarct size following MI.

<p>TNFR1 and TNFR2 gene deletion had opposite effects upon infarct size (A) and interstitial fibrosis in remote non-ischemic regions (B). N = 9–11/group. ^$P<0.05 vs. WT. All results were obtained from animals subjected to 7 days of MI.</p

Loss of GRK2 in Cardiomyocytes Decreases Cytochrome C Release from Mitochondria after I/R injury and Increases Levels of the Anti-apoptotic Bcl-2 proteins.

<p>(<b>A</b>) Representative Western blot of cytosolic levels of GRK2 and cytochrome C (CytoC) in myocardial lysates purified from MCM, GRK2fl/fl, GRK2iKO, and GRK2KO mice 30 min after I/R injury. (<b>B</b>) Mean±SEM of CytoC release in MCM and GRK2iKO mice under Sham conditions and post-I/R with n-sizes shown within the individual bars, *<i>p</i><0.05 MCM-I/R vs MCM-Sham, ^#<i>p</i><0.05 GRK2iKO-I/R vs. MCM-I/R mice. (<b>C</b>) Mean±SEM of CytoC release in GRK2fl/fl and GRK2KO mice under Sham conditions and post-I/R with n-sizes shown within the individual bars, *<i>p</i><0.05 GRK2fl/fl-I/R vs GRK2fl/fl-Sham, ^#<i>p</i><0.05 GRK2KO-I/R vs. GRK2fl/fl-I/R mice. (<b>D</b>) Representative Western blot showing GRK2, Bcl-2 and Bcl-xl protein levels in myocardial lysates purified from Sham or post-I/R GRK2fl/fl control mice or constitutive GRK2KO mice. (<b>E–F</b>) Quantitative levels (Mean±SEM) of BCL-2 (<b>E</b>) or BCL-xL (<b>F</b>) found in myocardial lysates from Sham or post-I/R GRK2fl/fl control mice or GRK2KO mice, *<i>p</i><0.05 KO vs GRK2fl/fl (n = 4 per group). (<b>G</b>) Representative Western blot showing GRK2, Bcl-2 and Bcl-xl protein levels in myocardial lysates purified from Sham or post-I/R MCM control mice or inducible GRK2iKO mice. (H-I) Quantitative levels (Mean±SEM) of BCL-2 (<b>H</b>) or BCL-xL (<b>I</b>) found in myocardial lysates from Sham or post-I/R MCM control mice or GRK2iKO mice, *<i>p</i><0.05 KO vs GRK2fl/fl (n = 4 per group).</p

GRK5W30A displays increased plasma membrane association following agonist treatment and differential ability to desensitize GPCRs compared to WT.

<p>(<b>A</b>) AdRbM were infected with an adenovirus expressing GRK5-GFP or GRK5W30A-GFP and cultured overnight. Using TIRFM cells were imaged at 10 sec intervals for 700 sec. Baseline myocytes were untreated while stimulated myocytes were treated with either AngII (10 µM) (A) or PE (50µM) (<b>B</b>) at 120 sec. Fluorescence was normalized and reported to fold change versus baseline. n = 4 (<b>C</b>) Changes in GRK5 activity at the membrane was measured using an IP₁ ELISA to determine changes in desensitization. NRVM were infected with Ad-LacZ, Ad-GRK5 or Ad-GRK5W30A. After 48 hours, cells were stimulated with PE or ET-1 for 2 hr, then assayed for IP₃ generation via IP₁ ELISA. *p<0.01 v. LacZ PE and WT PE, #p<0.01 v. LacZ ET-1, one-way ANOVA with a Bonferroni correction, n = 3, done in duplicate.</p