FADD^−/− ameliorates post-I/R myocardial infarct size.

<p>(A) Representative photograph of TTC stained heart tissue section obtained 24 hours I/R in FADD^−/− and NLC groups. (B) Graphic summery of infarct size expressed as percentage of area-at-risk (AAR) and the size of AAR. n = 8–10 mice/group. **<i>P</i><0.01, FADD^−/− vs. NLC control.</p

Effect of FADD^−/− reduced post-I/R cardiomyocyte apoptosis.

<p>(A) Representative photomicrographs of in situ detection of cardiac tissue DNA fragments from mice subjected to 30 minutes of ischemia and 3 hours or 7 days of reperfusion. Tissue sections were stained with DAPI (blue), anti-actinin (red) and TUNEL (green). TUNEL-positive nuclei were summarized in graph (B) and expressed as percentage of all tissues subject to I/R staining TUNEL-positive. (C) Caspase-3, -8, and -9 activity in ischemic cardiac tissue after 3 hours reperfusion. n = 10–12 animals/group. *<i>P</i><0.05, **<i>P</i><0.01 vs. NLC control.</p

Effect of FADD^−/− on cardiac infarct size, cardiac remodeling and survival after 6 weeks MI.

<p>(A) representative TTC stained heart tissue section at 6 week post-MI in FADD^−/− and NLC control groups. (B) Graphic summary of infarct size expressed as the length of the scare/LV circumference, n = 8 animals in each group. (C) Graphic presentation of LV area in NLC and FADD^−/− groups, <i>P</i><0.05 FADD^−/− vs. NLC, n = 8 in each groups. (D) Graphic presentation of LVIDd measured by echocardiography. <i>P</i><0.05 FADD^−/− vs. NLC, n = 8 in each groups. (E) Graphic presentation the ratio of heart length (HL)/tibia length (TL) in sham or MI mice. <i>P</i><0.05 FADD^−/− vs. NLC, n = 8 in each groups. (F) Survival curve in 8 week post-sham (n = 8 in each group) or post-MI mice (n = 23 in each group). *<i>P</i><0.05, FADD^−/− vs. NLC control.</p

Effect of FADD^−/− upon cardiac function as determined by ventricular catheterization, spanning pre- to 7 days post-reperfusion.

<p>(A) Left ventricular systolic pressure (LVSP). (B) Left ventricular end diastolic pressure (LVEDP). (C) Rate of rise of left ventricular pressure (+dP/dt) and (D) Rate of reduction of left ventricular pressure (−dP/dt). n = 10–14 mice/group. *<i>P</i><0.05 FADD^−/− vs. NLC control.</p

Effect of FADD^−/− upon cardiac function as determined by echocardiography.

<p>(A) Representative echocardiographic recordings pre- and post-24 hours and 7 days of reperfusion. (B) and (C) Graphic summary of LV ejection fraction (LVEF) and LV fractional shortening (LVFS) in groups (n = 10–14 mice/group). *<i>P</i><0.05, **<i>P</i><0.01 FADD^−/− vs. NLC control (FADD∶GFP MHC-Cre⁻).</p

Time course of FADD expression in each group post-myocardial ischemia/reperfusion (I/R).

<p>(A) Top: Representative photomicrographs of FADD expression in cardiac tissue by western blot in WT C57/BL6 (lanes 1–2) and FADD^+/−-FADD∶GFP-MHC-Cre⁻ (lanes 3–4) control mice, FADD^−/−-FADD∶GFP-MHC-Cre⁻ (NLC, line 5–6) and FADD^−/−-FADD∶GFP-MHC-Cre⁺ (FADD^−/−, line 7–8) mice. Bottom: Ratio of FADD expression (FADD or FADD+FADD∶GFP to GAPDH). (B) Top: Representative photomicrographs of FADD∶GFP expression in cardiac tissue pre- and post-MI/R by western blot in FADD^−/−-FADD∶GFP-MHC-Cre⁻ (NLC) and FADD^−/−-FADD∶GFP-MHC-Cre⁺ (FADD^−/−) groups. Bottom: Ratio of FADD expression (FADD∶GFP to GAPDH). n = 4. **<i>P</i><0.01 vs. FADD^−/−-FADD∶GFP-MHC-Cre⁻ (NLC). ^#<i>P</i><0.05 vs pre-I/R condition in NLC and FADD^−/− groups respectively, n = 4.</p

Knockdown FNDD in cell reduces chelerythrin-induced apoptosis.

<p>(A) Representative real time PCR tracings of transcript for 18S and FADD in H9C2 cells. Black: control, green: scramble siRNA, and red: FADD siRNA. (B) Quantification of relative mRNA expression of FADD in FADD specific siRNA or scrambled siRNA transfected H9C2 cells compared to control, *<i>P</i><0.05, <i>t</i> test, n = 4 per group. (C) Representative Western blot showing the release of cleaved caspase-3 (CC-3) in H9C2 cells treated with chelerythrin. (D) Quantification of CC-3 release in FADD specific siRNA or scrambled siRNA transfected H9C2 cells compared to that of control cells, *<i>P</i><0.05, ANOVA, n = 4 per group.</p

GRK5 nuclear accumulation is diminished after treatment with a CaM inhibitor.

<p>(<b>A</b>) NRVM were infected with Ad-GRK5 and either Ad-LacZ or Ad-Gq-CAM. 48 hr after infection, cells were treated with DMSO or inhibitor: BIM1 (10 µM), Gö6976 (10 µM), CDZ (10 µM) and KN93 (10 µM) for 1 hr. The cells were harvested using subcellular fractionation and immunoblotted for GRK5. (<b>B</b>) Immunoblots were quantitated by densitometry, normalized to fibrillarin, and reported as fold change over baseline. * p<0.05 v. untreated baseline, # p<0.05 v. CDZ, one-way ANOVA with a Bonferroni correction, n = 4. (<b>C</b>) NRVM were infected with Ad-LacZ, Ad-GRK5 and Ad-Gq-CAM. 48 hr after infection, cells were treated with DMSO or CDZ (10 µM) for 1 hr. The cells were harvested using subcellular fractionation, and immunoblotted for GRK5. (<b>D</b>) Densitometric analysis for (<b>C</b>) with GRK5 normalized to fibrillarin and calculated as fold change over baseline. *p<0.01 v. DMSO GRK5, #p<0.01 v. DMSO GRK5+ Gq, one-way ANOVA with a Bonferroni correction, n = 4. (<b>E</b>) NRVM were infected with either Ad-LacZ or Ad-Gq-CAM. 48 hr after infection, cells were treated with DMSO or CDZ (10 µM). Immunofluorescence was detected using a polyclonal GRK5 antibody. (<b>F</b>) TIRF analysis of AdRbM infected with an adenovirus expressing GRK5-GFP and cultured overnight. Cells were imaged at 10 sec intervals for 700 sec. Cells were pre-treated with CDZ or DMSO for 30 min at 37°C prior to imaging. Baseline myocytes were untreated while stimulated myocytes were treated with PE (50 µM) at 120 sec. Fluorescence was normalized and reported to fold change versus baseline. n = 4. (<b>G</b>) Same experimental design as (<b>F</b>) except cells were stimulated with AngII (10 µM) at 120 s. n = 4.</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

PE and AngII induce translocation of GRK5 from the membrane to the nucleus.

<p>(<b>A</b>) Representative immunofluorescence staining of endogenous GRK5 in AdRbM shows increased nuclear GRK5 following PE (50 µM) and AngII (10 µM) treatment, but not ET-1 (100 nM). (<b>B</b>) NRVM were infected with Ad-GRK5 (50 MOI). After 48 hr, cells were treated with 50 µM PE for 5 different time points, harvested by subcellular fractionation. Nuclear were fractions immunoblotted for GRK5 and fibrillarin. (<b>C</b>) The amount of GRK5 in the nucleus was calculated by denistometry, normalized to fibrillarin, and reported as Fold Change over baseline. *p<0.05, one-way ANOVA with a Bonferroni correction, n = 4. (<b>D</b>) Rabbit myocytes were infected with an adenovirus expressing GRK5-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 PE (50 µM), AngII (10 µM), or Et-1 (100 nM) at 120 sec. Fluorescence was normalized and reported as fold change versus baseline. n = 4. (<b>E</b>) Representative TIRF images for each agonist at the beginning and end of imaging.</p