Hexokinase II knockdown results in exaggerated cardiac hypertrophy via increased ROS production

Abstract

Hexokinase-II (HKII) is highly expressed in the heart and can bind to the mitochondrial outer membrane. Since cardiac hypertrophy is associated with a substrate switch from fatty acid to glucose, we hypothesized that a reduction in HKII would decrease cardiac hypertrophy after pressure overload. Contrary to our hypothesis, heterozygous HKII-deficient (HKII(+/-)) mice displayed increased hypertrophy and fibrosis in response to pressure overload. The mechanism behind this phenomenon involves increased levels of reactive oxygen species (ROS), as HKII knockdown increased ROS accumulation, and treatment with the antioxidant N-acetylcysteine (NAC) abrogated the exaggerated response. HKII mitochondrial binding is also important for the hypertrophic effects, as HKII dissociation from the mitochondria resulted in de novo hypertrophy, which was also attenuated by NAC. Further studies showed that the increase in ROS levels in response to HKII knockdown or mitochondrial dissociation is mediated through increased mitochondrial permeability and not by a significant change in antioxidant defenses. Overall, these data suggest that HKII and its mitochondrial binding negatively regulate cardiac hypertrophy by decreasing ROS production via mitochondrial permeability.

Figure 1. Cardiac hypertrophic response in HKII^+/− hearts after TAC

1. HW/TL ratio in WT and HKII^+/− mice subjected to TAC for 2 and 4 weeks as indicated, *p < 0.0001 versus WT sham at baseline, ^#p = 0.02 versus WT + TAC (2W) and ^#p < 0.0001 versus WT + TAC (4W) and n = 8–12.

2. Representative gross images of hearts after sham or TAC operation for 4 weeks from WT and HKII^+/− mice.

3. Haematoxylin and eosin (H&E) staining of TAC operated WT and HKII^+/− mice at 4 weeks. Quantification of myocyte size from cardiac histological sections of WT and HKII^+/− heart 4 weeks after TAC is shown on the bottom, *p < 0.0001 versus WT sham, ^#p < 0.0001 versus WT + TAC and n > 400 cells from three animals in each group.

4. Representative M-mode images of WT and HKII^+/− hearts after 4 weeks of TAC. LVED septum wall thickness is shown on the bottom, red lines indicate interventricular septal wall thickness, *p < 0.0001 versus WT sham, ^#p = 0.008 versus WT + TAC and n = 8–10.

5. Real-time PCR analysis of ANF levels from WT and HKII^+/− hearts 2 and 4 weeks after TAC, *p < 0.001 versus WT sham at baseline, ^#p = 0.005 versus WT + TAC and n = 5.

6. Real-time PCR analysis of BNP levels, *p = 0.010 versus WT sham at baseline, *p < 0.0001 HKII^+/− versus WT sham at baseline, ^#p = 0.042 versus WT + TAC (4W) and n = 5. ANOVA was performed for all experimental conditions and data are presented as mean ± SEM.

Figure 2. Transition from hypertrophy to heart failure after TAC

1. Histological view of cardiac sections stained with Masson's trichrome in WT and HKII^+/− mice after 8 weeks of TAC. Quantification of fibrosis is shown next to the image, *p = 0.035 versus WT and n = 3. Scale bar = 50 µm.

2. Quantification of TUNEL staining in the hearts of WT and HKII^+/− mice after 8 weeks of TAC, *p < 0.0001 versus WT sham, ^#p = 0.01 versus WT + TAC and n = 3.

3. Kaplan–Meier survival curve of WT and HKII^+/− mice subjected to TAC for 8 weeks, ^#p = 0.032, n = 35 WT, n = 29 HKII^+/− and log-rank test.

4. EF% (ejection fraction) in WT and HKII^+/− after 4 and 8 weeks of TAC as assessed by echocardiography, *p < 0.0001 versus WT sham at baseline, ^#p = 0.048 versus WT + TAC (4W), ^#p = 0.008 versus WT + TAC (8W) and n = 8–12.

5. FS% (fractional shortening) in WT and HKII^+/− after 4 and 8 weeks of TAC as assessed by echocardiography, *p = 0.0002 versus WT sham at baseline (HKII^+/− + TAC 4W), *p < 0.0001 versus WT sham at baseline (TAC 8W), ^#p = 0.004 versus WT + TAC (8W) and n = 8–12.

6. LVEDD in the hearts of WT and HKII^+/− mice after 8 weeks of TAC, *p < 0.0001 versus WT sham; ^#p = 0.003 versus WT + TAC and n = 8–10.

7. Lung weight to body weight ratio (LW/BW) in WT and HKII^+/− subjected to TAC for 8 weeks, *p = 0.016 versus WT sham, *p < 0.0001 HKII^+/− versus WT sham, ^#p < 0.0001 versus WT + TAC and n = 4–6. ANOVA was performed for all experimental conditions unless otherwise indicated, and data presented as mean ± SEM.

Figure 3. Hypertrophic response to AngII in NRCM

1. Western blot of total HKII levels in NRCM treated with AngII (250 nM, 0–12 h), *p = 0.045, n = 3 and two-tailed t-test.

2. In (B–E), NRCM were treated with scrambled or HKII siRNA for 36 h prior to media change and treatment with AngII (250 nM, 24 h). Representative images and cell size analysis of cardiomyocytes immunostained with sarcomeric α-actinin (red). Data represent the total cardiomyocyte area relative to untreated siRNA control from at least three-independent samples, a minimum of 75 total cells per group, *p = 0.001 siCtrl + AngII versus siCtrl, *p < 0.0001 siHKII + AngII versus siCtrl, ^#p = 0.037 versus siCtrl + AngII and two-tailed t-test. DAPI (blue) was used as a nuclear counterstain, and scale bar: 20 µm.

3. Real-time PCR analysis of ANF expression ± AngII. Data represent average mRNA levels relative to the untreated control for each group, *p = 0.010 siCtrl + AngII versus siCtrl, *p = 0.001 siHKII + AngII versus siCtrl, ^#p = 0.049 versus siCtrl + AngII, n = 4–6 and two-tailed t-test.

4. Real-time PCR analysis of BNP expression ± AngII. Data represent average mRNA levels relative to the untreated control for each group, *p = 0.016 siCtrl + AngII versus siCtrl, *p = 0.001 siHKII + AngII versus siCtrl, ^#p = 0.009 versus siCtrl + AngII, n = 4–6 and two-tailed t-test.

5. Protein synthesis, evaluated by [³H]-leucine incorporation into total cellular protein normalized to cell number. Data are presented relative to untreated siRNA control, *p = 0.011 siCtrl + AngII versus siCtrl, *p = 0.0002 siHKII + AngII versus siCtrl, ^#p = 0.014 versus siCtrl + AngII, n = 4–6 and two-tailed t-test. All data are presented as mean ± SEM.

Figure 4. ROS accumulation in response to HKII knockdown

1. Analysis of malondialdehyde (MDA) levels in left ventricular tissue from HKII^+/− mice compared to WT 2 days after TAC, *p = 0.049 versus WT sham, *p = 0.001 HKII versus WT sham, ^#p = 0.009 versus WT + TAC and n = 3–5.

2. ROS levels in NRCM, evaluated by Western blot of derivitized protein carbonyls (DNP) in lysates from NRCM treated with HKII or control siRNA ± AngII (250 nM, 24 h). All represented lanes were run non-contiguous on the same gel. Data are presented relative to untreated siRNA control, *p = 0.013 siCtrl + AngII versus siCtrl, *p = 0.002 siHKII + AngII versus siCtrl, ^#p = 0.046 versus siCtrl + AngII, n = 3 and two-tailed t-test.

3. ROS levels in NRCM evaluated with DCF (green). Fluorescent intensity was measured in individual fields from 3 to 6 independent samples and presented relative to control, *p = 0.014 siCtrl + AngII versus siCtrl, *p = 0.0006 siHKII + AngII versus siCtrl, ^#p = 0.034 versus siCtrl + AngII and two-tailed t-test.

4. Mitochondrial superoxide evaluated with MitoSox (red). Fluorescent intensity was averaged from 3 to 6 independent samples (three fields analysed per sample), *p = 0.0004 siCtrl + AngII versus siCtrl, *p = 0.0002 siHKII + AngII versus siCtrl, ^#p = 0.006 versus siCtrl + AngII and two-tailed t-test. In (C) and (D), nuclei are labelled with Hoechst 33342 (blue), scale bars: 20 µM. All data are presented as mean ± SEM.

Figure 5. Hypertrophic response after treatment with the antioxidant NAC

1. In (A–D), NRCM were treated with scrambled or HKII siRNA, and exposed to AngII (250 nM, 24 h). NAC (1 mM) was added to the media 2 h after the addition of AngII where indicated. Representative images and cell size analysis of cardiomyocytes immunostained with sarcomeric α-actinin (red). Data represent the total cardiomyocyte area relative to untreated siRNA control from at least three-independent samples, a minimum of 75 total cells per group, *p = 0.037 versus siCtrl + AngII, ^#p = 0.010 versus siCtrl + AngII, ^#p = 0.014 versus siHKII + AngII and two-tailed t-test. DAPI (blue) was used as a nuclear counterstain and scale bar: 20 µm.

2. Real-time PCR analysis of ANF levels. Data represent average mRNA levels for each marker relative to the untreated siRNA control for each group, *p = 0.015 versus siCtrl + AngII, ^#p = 0.004 versus siHKII + AngII, n = 3–6 and two-tailed t-test.

3. Real-time PCR analysis of BNP levels. Data represent average mRNA levels for each marker relative to the untreated siRNA control for each group, *p = 0.016 versus siCtrl + AngII, ^#p = 0.035 versus siCtrl + AngII, ^#p = 0.020 versus siHKII + AngII, n = 3–6 and two-tailed t-test.

4. Protein synthesis evaluated by [³H]-leucine incorporation into total cellular protein normalized to cell number. [³H]-leucine (1 µCi/ml) was added to cells to cells 1 h after treatment with NAC. Data are presented relative to untreated siRNA control, *p = 0.049 versus siCtrl + AngII, ^#p = 0.015 versus siCtrl + AngII, ^#p = 0.001 versus siHKII + AngII, n = 4–6 and two-tailed t-test. All data are presented as mean ± SEM.

Figure 6. Hypertrophic response to HKII dissociation from the mitochondria

1. Western blot demonstrating HKII levels in mitochondrial fractions from WT and HKII^+/− hearts 4 weeks after TAC, *p < 0.0001 versus sham, ^#p < 0.0001 versus HKII^+/− + TAC, n = 4–6 and determined by ANOVA. Mitochondrial inner membrane protein succinate dehydrogenase (SDH) was used as a loading control.

2. Western blot of HKII levels in mitochondrial fractions from NRCM treated with AngII (200 nM, 6 h). Represented lanes for each Western blot were run non-contiguous on the same gel. Data are presented relative to untreated control, *p = 0.049, n = 3 and two-tailed t-test.

3. Mitochondrial superoxide levels evaluated with MitoSox (red) after HKII dissociation from the mitochondria in NRCM using a competitive peptide (n-HKII) (5 µM, 3 h). A scrambled peptide (scram) was used as control. Cells were pretreated with NAC (500 µM) where indicated. Fluorescent intensity was measured from at least three-independent experiments, *p = 0.011 versus scram, ^#p = 0.019 versus n-HKII and two-tailed t-test.

4. In (D–F), NRCM were treated with n-HKII or scrambled peptide (5 µM, 24 h), and pretreated with NAC (500 µM) where indicated. Representative images and cell size analysis of cardiomyocytes. Data represent the total cardiomyocyte area relative to scrambled peptide control from at least three-independent samples, a minimum of 75 total cells per group, *p = 0.001 versus scram and ^#p = 0.025 versus n-HKII. DAPI (blue) was used as a nuclear counterstain and scale bar: 20 µm.

5. Real-time PCR analysis of ANF expression. Data are presented relative to scrambled control, *p = 0.004 versus scram, ^#p = 0.041 versus n-HKII, n = 4–6 and two-tailed t-test.

6. Protein synthesis, evaluated by [³H]-leucine incorporation and normalized to cell number. Data are presented relative to scrambled control, *p = 0.005 versus scram, ^#p = 0.007 versus n-HKII, n = 3 and two-tailed t-test. All data are presented as mean ± SEM.

Figure 7. Assessment of MPT after HKII knockdown or mitochondrial dissociation

1. Representative images of calcein distribution (green) after knockdown and treatment with AngII (250 nM, 2 h). NAC (500 µM) was added after 20 min where indicated. Mitochondrial are labeled with Mitotracker Red; scale bar: 20 µM.

2. Quantification of cells demonstrating calcein release from the mitochondria after knockdown and treatment with AngII. A minimum of 150 total cells in each group were analysed from at least three-independent experiments. Data represent the percentage of cells with released calcein relative to untreated siRNA control, *p = 0.016 siCtrl + AngII versus siCtrl, *p = 0.002 siHKII + AII versus siCtrl, ^#p = 0.041 versus siCtrl + AngII, **p = 0.016 versus siCtrl + AngII and ^##p =.001 versus siHKII + AngII.

3. Representative images of calcein distribution after treatment with the n-HKII dissociation peptide or the scrambled (scram) control (5 µM, 2 h). Cells were pretreated with NAC (10 min, 500 µM) where indicated. Mitochondrial are labeled with Mitotracker Red; scale bar: 20 µM.

4. Quantification of cells demonstrating calcein release from the mitochondria after peptide treatment. A minimum of 145 total cells in each group were analysed from at least three-independent experiments. Data represent the percentage of cells with released calcein relative to scrambled control, *p = 0.005 versus scram and ^#p = 0.008 versus n-HKII. All data are presented as mean ± SEM.