Reduction in hexokinase II levels results in decreased cardiac function and altered remodeling after ischemia/reperfusion injury

Abstract

Rationale: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane.

Objective: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R.

Methods and results: We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII(+/)⁻) mice to I/R by coronary ligation. At baseline, HKII(+/)⁻ mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor-dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O₂ consumption, and contractility.

Conclusions: These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.

Figure 1

Oxidative stress and I/R reduce HKII-mitochondrial binding. Mitochondria-bound (A) and total HKII levels (B) in NRCM treated with and without H₂O₂ or doxorubicin. n = 3 independent experiments. (C and D) Mitochondria-bound and total HKII levels in wild type and HKII^+/− mice 2 days after I/R injury and in sham-operated mice. *P < 0.05, n = 4–6 samples. Data are presented as mean ± SEM.

Figure 2

HKII^+/− mice display lower cardiac function after I/R. Cardiac function was assessed via Doppler echocardiography and by hemodynamic analysis. (A) Summary of FS measurements in WT and HKII^+/− mice at baseline and 2, 14, and 28 days after I/R. (B) dP/dt after sham operation or 28 days after I/R based on hemodynamic measurements from a pressure-volume loop in WT and HKII^+/− mice. (C) Left ventricular end diastolic pressure (LVEDP) in WT and HKII^+/− mice 28 days after I/R. (D) −dP/dt in WT and HKII^+/− mice. *P < 0.05. n = 8–10 animals. Data are presented as mean ± SEM.

Figure 3

HKII^+/− hearts have lower function and increased cell death after I/R ex vivo. HKII^+/− hearts display a higher degree of LDH release and cell injury (A), no change in the rate pressure product (B), a higher end diastolic pressu re (C), and no change in DLVP (D). LDH release and RPP were measured during 60 min reperfusion following 40 min ischemia. EDP was assessed at baseline and after 60 minutes of reperfusion. *P < 0.05, n = 6. Data are presented as mean ± SEM.

Figure 4

HKII^+/− mice display increased cell death and fibrosis after I/R. (A) Summary of area at risk (AAR) per left ventricle (LV) and infarct area (Inf) per AAR 2 days after I/R in WT and HKII^+/− hearts. (B) H&E stain of the peri-infarct area demonstrates more cellular damage in the HKII^+/− than WT hearts 2 days after I/R. (C) Histological images stained with TUNEL (green), phalloidin-Alexa for actin antibody (red), and DAPI for nuclei (blue) of the peri-infarct zone 2 days after I/R or sham operation in hearts from WT and HKII^+/− mice. (D) Summary of TUNEL stain studies. * P < 0.05 vs sham; # P < 0.05 vs WT I/R group. (E) Western blot of cytosolic cytochrome c levels 2 days after I/R in the WT and HKII^+/− hearts. A summary of the results is shown below the western blot. (F) Upper panel shows gross examination of WT and HKII^+/− hearts 28 days after coronary ligation, and the middle panel shows histological images stained with Masson’s Trichrome. Analysis of fibrosis determined by the ratio of fibrosis length over left ventricle (LV) circumference is shown in the lower panel. *P < 0.05, n = 6. Scale bar = 100 μM. Data are presented as mean ± SEM.

Figure 5

HKII^+/− mice have less angiogenesis in the peri-infarct zone. (A) Angiogenesis, as assessed by lectin antibody staining, is significantly reduced in the border zone of HKII^+/− hearts 28 days after I/R. Green areas represent lectin-positive cells and blue is DAPI for nuclei. Summary of the angiogenesis data is shown next to the images. Five representative high power fields (x40) from the peri-infarctarea of each section were examined, and the number of capillaries was calculated. (B) VEGF mRNA levels in WT and HKII^+/− mouse heart 28 days after I/R or sham operation. (C) HIF1α protein levels in WT and HKII^+/− mouse heart 2 days after I/R. Band intensity was normalized to tubulin first and then to WT. (D) Western blot of HIF1α in NRCM treated with control or HKII siRNA under hypoxic conditions. The bar graph below the blot represents a summary of the results. Band intensities were normalized to control siRNA. (E) Luciferase assay results on H9c2 cells transfetced with HRE-luciferase or control vector and treated with control or HKII siRNA and then cultured under normoxic and hypoxic conditions. * P < 0.05, **P < 0.01, n ≥ 3 in each group. Scale bar = 100 μM. Data are presented as mean ± SEM.

Figure 6

Displacement of endogenous HKII from mitochondria increases cell death. (A) Compared to a control peptide, 2 hour treatment with 5 μM of an HKII-competing peptide (n-HKII) resulted in a non-significant reduction in the amount of mitochondrially bound HKII in NRCM (left), while20 μM concentration resulted in almost complete displacement of HKII from the mitochondria (right). (B) Treatment with the n-HKII peptide for 2 hours leads to a dose dependent decrease in the mitochondrial membrane potential, as measured by flow analysis of TMRE fluorescence. (C) Treatment with the n-HKII peptide for 2 hours leads to a dose dependent increase in cell death compared to control, as assessed by trypan blue exclusion studies. *P < 0.05 vs control peptide, n = 3 in each group. Data are presented as mean ± SEM.

Figure 7

HKII overexpression increases ATP production and O₂ consumption, while its downregulation has the opposite effect. (A) HKII was overexpressed in NRCM using an adenovirus and ATP levels were measured. Cells overexpressing HKII displayed significantly higher ATP levels compared to cells overexpressing GFP alone. (B) NRCM were treated with control or HKII siRNA and ATP levels were measured after 48 hours. HKII downregulation resulted in a reduction in cellular ATP levels when normalized to cell number. (C) Summary of baseline and maximal O₂ consumption in NRCM treated with GFP and HKII adenovirus. HKII overexpression resulted in a higher maximal O₂ consumption in NRCM compared to GFP transfected live cells. (D) HKII downregulation resulted in a decrease in both basal and maximal O₂ consumption. *P < 0.05, n ≥ 3 experiments. Data are presented as mean ± SEM.