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. 2012 Apr;32(4):924-33.
doi: 10.1161/ATVBAHA.112.245134. Epub 2012 Mar 1.

Targeted focal adhesion kinase activation in cardiomyocytes protects the heart from ischemia/reperfusion injury

Zhaokang Cheng  1 , Laura A DiMichele, Zeenat S Hakim, Mauricio Rojas, Christopher P Mack, Joan M Taylor
Affiliations

Targeted focal adhesion kinase activation in cardiomyocytes protects the heart from ischemia/reperfusion injury

Zhaokang Cheng et al. Arterioscler Thromb Vasc Biol. 2012 Apr.

Abstract

Objective: We previously reported that cardiac-restricted deletion of focal adhesion kinase (FAK) exacerbated myocyte death following ischemia/reperfusion (I/R). Here, we interrogated whether targeted elevation of myocardial FAK activity could protect the heart from I/R injury.

Methods and results: Transgenic mice were generated with myocyte-specific expression of a FAK variant (termed SuperFAK) that conferred elevated allosteric activation. FAK activity in unstressed transgenic hearts was modestly elevated, but this had no discernable effect on anabolic heart growth or cardiac function. Importantly, SuperFAK hearts exhibited a dramatic increase in FAK activity and a reduction in myocyte apoptosis and infarct size 24 to 72 hours following I/R. Moreover, serial echocardiography revealed that the transgenic mice were protected from cardiac decompensation for up to 8 weeks following surgery. Mechanistic studies revealed that elevated FAK activity protected cardiomyocytes from I/R-induced apoptosis by enhancing nuclear factor-κB (NF-κB)-dependent survival signaling during the early period of reperfusion (30 and 60 minutes). Moreover, adenoviral-mediated expression of SuperFAK in cultured cardiomyocytes attenuated H(2)O(2) or hypoxia/reoxygenation-induced apoptosis, whereas blockade of the NF-κB pathway using a pharmacological inhibitor or small interfering RNAs completely abolished the beneficial effect of SuperFAK.

Conclusions: Enhancing cardiac FAK activity attenuates I/R-induced myocyte apoptosis through activation of the prosurvival NF-κB pathway and may represent a novel therapeutic strategy for ischemic heart diseases.

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Figures

Figure 1
Figure 1. Generation of cardiomyocyte-specific SuperFAK transgenic mice
(A) COS7 cells were non-transfected (NT), transfected with LacZ control or SuperFAK (SF) and grown to confluence (adherent) or re-plated on fibronectin (FN)-coated dishes for 30 min. Western blotting was performed with indicated antibodies. (B) Levels of basal myocardial FAK activity in 1-month-old SuperFAK transgenic (SF1 and SF2) and non-transgenic (NTG) mice. (C) Detection of flag-tagged SuperFAK in various tissues revealed myocardial-restricted expression in SF2 mice. (D) SF2 mice exhibit elevated levels of FAK activity during development.
Figure 2
Figure 2. Targeted FAK activation provided protection from I/R injury
Non-transgenic (NTG) littermate control and SF2 mice at 8–12 weeks of age were subjected to 30min of ischemia followed by reperfusion. (A) Representative heart slices at 24h or 72h post I/R stained with Evans blue/TTC showing infarct area (Evans blue and TTC unstained, white) and area at risk (Evans blue unstained, shown as red and white). (B) Infarct size (ratio of infarct area to area at risk) was significantly decreased in the SF2 mice compared with NTG mice 24h (NTG, n=6; SF2, n=7) or 72h (NTG, n=5; SF2, n=4) post I/R. * p < 0.05; *** p < 0.001. (C,D) LV ejection fraction (EF) and fractional shortening (FS) were evaluated by conscious echocardiography before, 2, 4, and 8 weeks post I/R. NTG, n=6; SF2, n=5. * p < 0.05 vs. NTG; ** p < 0.01 vs. NTG; *** p < 0.001 vs. NTG. Data are expressed as mean ± SEM.
Figure 3
Figure 3. FAK was activated in hypoxic cardiomyocytes and myocyte FAK activation conferred protection from I/R induced apoptosis
(A) SF2 mice were injected with 60 mg/kg (i.p.) hypoxyprobe-1 and sacrificed at 24h post I/R or sham operation. Heart sections were stained with mouse anti-hypoxyprobe-1 antibody to label hypoxic cells (red), and rabbit anti-pFAK (Y397) to show FAK activation (green). Signals for hypoxyprobe-1 and pFAK (Y397) were dramatically increased and co-localized (yellow-orange) following I/R compared with sham-operated SF2 hearts, indicative of FAK activation in hypoxic myocardial cells. Nuclei were labeled with DAPI (blue). Scale bar = 20µm. (B) At 24h post I/R, the ischemic border zone of SF2 hearts displayed elevated FAK activity (green) and decreased TUNEL staining (red) when compared with non-transgenic (NTG) littermate control hearts. Nuclei were labeled with DAPI (blue) and cardiomyocytes labeled with cardiac troponin T (cTnT, white). Scale bar = 20µm. (C) Quantification of TUNEL-positive myocytes in the infarct border (left) and remote (right) region showed significantly less apoptotic cardiomyocytes in the SF2 hearts than in the control hearts. NTG, n=3; SF2, n=4 (100–200 cells/heart). * p < 0.05 vs. NTG; ** p < 0.01 vs. NTG. Data are expressed as mean ± SEM.
Figure 4
Figure 4. Overexpression of SuperFAK protected cultured cardiomyocytes from oxidative stress-induced apoptosis
(A) Quantification of TUNEL-positive myocyte nuclei showing a significant decrease of apoptosis following H2O2 treatment in SuperFAK-infected neonatal rat cardiomyocytes (NRCM) when compared with non-infected or GFP-infected NRCM. Results are mean ± SEM of 4 independent experiments. * p < 0.05. (B) Non-infected, GFP- or SuperFAK-infected NRCM were subjected to hypoxia for 2h followed by re-oxygenation for 1h. Cellular apoptosis assessed by TUNEL staining was significantly decreased by overexpression of SuperFAK compared with intact or GFP-infected cells. Data represent mean ± SEM of 3 independent experiments. * p < 0.05.
Figure 5
Figure 5. FAK enhanced nuclear translocation and transcriptional activation of NF-κB in cardiac myocytes
(A–D) Non-transgenic (NTG) littermate control mice and SF2 mice were subjected to 30min of ischemia followed by 30min or 60min of reperfusion. Border zone of the infarcted myocardium was collected for subcellular fractionation or immunohistochemistry. (A) Representative immunoblots of whole cell lysates 30min and 60min post I/R with GAPDH as a loading control. (B) Quantification of protein expression in whole cell lysate at 60min post I/R showed a significant increase of phospho-NF-κB p65 (S536), phospho-IκBα (S32/36) and a decrease of total IκBα in the SF2 group when compared with control hearts. NTG, n=3; SF2, n=4. * p < 0.05 vs. NTG. (C) Representative immunoblots of nuclear and cytosolic fractions 30min and 60min post I/R. Histone H3 served as a nuclear loading control and GAPDH as a cytosolic loading control. See supplemental figure XII for quantification. (D) Confocal images of NTG and SF2 hearts 60min post I/R immunolabeled for NF-κB p65 (green), cardiac troponin T (cTnT, red) and DAPI (blue). SF2 hearts exhibited increased NF-κB p65 signal in the nuclei of cardiac myocytes (arrows). Scale bar = 20µm. (E) Protein levels of XIAP, Bcl-2 and Bcl-xl were significantly higher in the SF2 heart than in controls at 24h post I/R. See supplemental figure XII for Western images. NTG, n=3; SF2, n=4. * p < 0.05 vs. NTG. ** p < 0.01 vs. NTG. Data are mean ± SEM.
Figure 6
Figure 6. Blockade of NF-κB pathway abrogated the anti-apoptotic effect of FAK activation
(A) Neonatal rat cardiomyocytes (NRCM) overexpressing GFP or SuperFAK were treated with DMSO or the NF-κB inhibitor JSH-23 (25µM) for 12h. Cell lysates were probed with indicated antibodies by Western blotting, with GAPDH as a loading control. SF, SuperFAK. (B,C) NRCM were infected with adenoviruses overexpressing GFP or SuperFAK for 48h. Cells were then incubated with DMSO or the NF-κB inhibitor JSH-23 (25µM) for 2h before stimulation with H2O2 (B) or hypoxia/re-oxygenation (C) as described in Methods. TUNEL staining revealed that overexpression of SuperFAK significantly decreased myocyte apoptosis in response to oxidative stress. However, inhibition of NF-κB completely abolished the protective effect of SuperFAK. Data represent mean ± SEM of 4 independent experiments (200–400 cells/experiment). * p < 0.05. (D) NRCM were transfected with control siRNA (si-Control) or NF-κB p65 siRNA (si-p65) for 24h followed by infection with GFP or SuperFAK adenoviruses for 48h. Western blotting showed >90% knockdown of NF-κB p65. Images are representatives of 3 independent experiments. (E,F) NRCM in (D) were treated with H2O2 (E) or hypoxia/re-oxygenation (F). TUNEL staining revealed that knockdown of NF-κB completely abolished the protective effect of SuperFAK. Data represent 4 independent experiments. * p < 0.05.
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