Inhibition of Fas-associated death domain-containing protein (FADD) protects against myocardial ischemia/reperfusion injury in a heart failure mouse model

AIM As technological interventions treating acute myocardial infarction (MI) improve, post-ischemic heart failure increasingly threatens patient health. The aim of the current study was to test whether FADD could be a potential target of gene therapy in the treatment of heart failure. METHODS Cardiomyocyte-specific FADD knockout mice along with non-transgenic littermates (NLC) were subjected to 30 minutes myocardial ischemia followed by 7 days of reperfusion or 6 weeks of permanent myocardial ischemia via the ligation of left main descending coronary artery. Cardiac function were evaluated by echocardiography and left ventricular (LV) catheterization and cardiomyocyte death was measured by Evans blue-TTC staining, TUNEL staining, and caspase-3, -8, and -9 activities. In vitro, H9C2 cells transfected with ether scramble siRNA or FADD siRNA were stressed with chelerythrin for 30 min and cleaved caspase-3 was assessed. RESULTS FADD expression was significantly decreased in FADD knockout mice compared to NLC. Ischemia/reperfusion (I/R) upregulated FADD expression in NLC mice, but not in FADD knockout mice at the early time. FADD deletion significantly attenuated I/R-induced cardiac dysfunction, decreased myocardial necrosis, and inhibited cardiomyocyte apoptosis. Furthermore, in 6 weeks long term permanent ischemia model, FADD deletion significantly reduced the infarct size (from 41.20 ± 3.90% in NLC to 26.83 ± 4.17% in FADD deletion), attenuated myocardial remodeling, improved cardiac function and improved survival. In vitro, FADD knockdown significantly reduced chelerythrin-induced the level of cleaved caspase-3. CONCLUSION Taken together, our results suggest FADD plays a critical role in post-ischemic heart failure. Inhibition of FADD retards heart failure progression. Our data supports the further investigation of FADD as a potential target for genetic manipulation in the treatment of heart failure

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart

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