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^−/− 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

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

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

GRK2 Knock-down in Cultured Cardiomyocytes Reduces Cleaved Caspase 3 Levels and Increases Activated Akt and Bcl-2 Levels.

<p>(<b>A</b>) Representative Western blot for GRK2, total Akt, phospho-Akt (as in Fig. 7), Bcl-2, BCl-xL, cleaved caspase-3 and GAPDH as a loading control cultured neonatal rat ventricular myocytes (NRVMs) following 3 day treatment with control or GRK2-specific siRNA (see Methods) under basal conditions or after a challenge with Chelerythrine (Chele, 10 µM). (<b>B</b>) Quantitative assessment of GRK2 levels in NRVMs 3 days after siRNA treatment (control or GRK2 specific siRNA), *<i>p</i><0.05 GRK2-siRNA vs. Control-siRNA (ANOVA, n = 4 per group). (<b>C–F</b>) Quantitative assessment of protein levels of phosphor-Akt normalized to total Akt (<b>C</b>); Bcl-2 (<b>D</b>); Bcl-xl (<b>E</b>); and cleaved caspase-3 (<b>F</b>) from the corresponding NRVMs, *<i>p</i><0.05 between groups as indicated in each graph, n = 4.</p

Cardiac GRK2 KO Mice have Reduced Post-I/R Myocardial Apoptosis.

<p>(<b>A</b>) Representative photomicrographs of in situ detection of DNA fragments (TUNEL) in myocardial sections from Sham or post-I/R mice from the listed groups (MCM and GRK2fl/fl as controls as GRK2iKO and GRK2KO). Total nuclei were labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining (green) and a-actin labeled myocytes in red. (<b>B,C</b>) Mean±SEM of the percentage of TUNEL positive nuclei per total cell number per field (<b>B</b>) and percentage of TUNEL positive myocytes per field (<b>C</b>), *<i>p</i><0.05 vs. MCM and GRK2fl/fl control groups (ANOVA). (<b>D–F</b>) Activity (mean±SEM) of caspase-3 (<b>D</b>), -8 (<b>E</b>), and -9 (<b>F</b>) in all groups with n-sizes per group listed at bottom of graphs, *<i>p</i><0.05 vs. MCM and GRK2fl/fl control groups.</p