Glucose transporter 4-deficient hearts develop maladaptive hypertrophy in response to physiological or pathological stresses

Glucose transporter 4 (GLUT4) is required for myocardial adaptations to exercise, and its absence accelerates heart dysfunction after pressure overload. The requirement for GLUT4 may extend beyond glucose uptake to include defects in mitochondrial metabolism and survival signaling pathways that develop in its absence. Therefore, GLUT4 is critical for responses to hemodynamic stresses.

Abstract

Pathological cardiac hypertrophy may be associated with reduced expression of glucose transporter 4 (GLUT4) in contrast to exercise-induced cardiac hypertrophy, where GLUT4 levels are increased. However, mice with cardiac-specific deletion of GLUT4 (G4H^−/−) have normal cardiac function in the unstressed state. This study tested the hypothesis that cardiac GLUT4 is required for myocardial adaptations to hemodynamic demands. G4H^−/− and control littermates were subjected to either a pathological model of left ventricular pressure overload [transverse aortic constriction (TAC)] or a physiological model of endurance exercise (swim training). As predicted after TAC, G4H^−/− mice developed significantly greater hypertrophy and more severe contractile dysfunction. Somewhat surprisingly, after exercise training, G4H^−/− mice developed increased fibrosis and apoptosis that was associated with dephosphorylation of the prosurvival kinase Akt in concert with an increase in protein levels of the upstream phosphatase protein phosphatase 2A (PP2A). Exercise has been shown to decrease levels of ceramide; G4H^−/− hearts failed to decrease myocardial ceramide in response to exercise. Furthermore, G4H^−/− hearts have reduced levels of the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1, lower carnitine palmitoyl-transferase activity, and reduced hydroxyacyl-CoA dehydrogenase activity. These basal changes may also contribute to the impaired ability of G4H^−/− hearts to adapt to hemodynamic stresses. In conclusion, GLUT4 is required for the maintenance of cardiac structure and function in response to physiological or pathological processes that increase energy demands, in part through secondary changes in mitochondrial metabolism and cellular stress survival pathways such as Akt.

NEW & NOTEWORTHY Glucose transporter 4 (GLUT4) is required for myocardial adaptations to exercise, and its absence accelerates heart dysfunction after pressure overload. The requirement for GLUT4 may extend beyond glucose uptake to include defects in mitochondrial metabolism and survival signaling pathways that develop in its absence. Therefore, GLUT4 is critical for responses to hemodynamic stresses.

INTRODUCTION

Cardiac hypertrophy can be induced by increased workload from physiological stresses (e.g., exercise) or from pathological stresses (e.g., hypertension) (1). In the case of exercise, a compensated hypertrophy develops with preserved contractile function and increased mitochondrial oxidative capacity (9, 30). However, when the heart is subjected to chronic pressure overload, there is a gradual decline in mitochondrial function resulting in decreased fatty acid oxidation (FAO) and a concurrent increased reliance on glycolysis associated with cardiac dilation and loss of contractility (26, 31). Given the potential role of changes in mitochondrial metabolic substrate utilization in the failing heart, a number of studies have focused on the contribution of altered substrate delivery. The solute carrier 2A (Slc2a) family of genes encode glucose transporter (GLUT) proteins (16). Of the 12 GLUTs conserved in mammals, GLUT1, GLUT4, and GLUT8 are the most abundant in the heart (3). In both human heart failure and mouse models of heart failure, GLUT4 expression is decreased, whereas GLUT1 expression is increased (36). We recently reported that in response to surgically induced pressure overload, loss of GLUT1 does not accelerate the transition from compensated hypertrophy to heart failure (26). In a separate study, when GLUT1 expression was transgenically induced in the adult heart and subjected to the same pressure overload model, mitochondrial dysfunction was prevented, and structural remodeling was attenuated (25), supporting earlier work indicating a protective role for chronic GLUT1 induction against pressure overload in mice (19). In addition, cardiomyocyte-restricted GLUT4 knockout (G4H^−/−) mice have a mild cardiac hypertrophy with preserved contractile function in the unstressed state (2). However, when hearts from these mice are subjected to ischemic injury, they have delayed recovery that is further exacerbated by metabolically stressing the mice with food deprivation (37). Together, these studies in mouse models of altered expression of either GLUT1 or GLUT4 support the hypothesis that GLUT4 may be required for an adaptive cardiac response to increased hemodynamic demands.

To determine whether GLUT4 expression is required for functional, structural, and metabolic adaptations to hemodynamic stress, we subjected G4H^−/− and control (Con) littermates to either intermittent hemodynamic stress by swim training or chronic pressure overload induced by transverse aortic constriction (TAC). Our data show that GLUT4 is required for cardiac responses to either a physiological intermittent stress or a pathological chronic stress.

MATERIALS AND METHODS

Experimental mice.

Mice with cardiomyocyte-restricted deletion of GLUT4 were previously generated using the Cre-loxP system (2). Briefly, mice with homozygous floxed Glut4 alleles in which loxP sites flank exon 10 of the Glut4 gene (Glut4^lox/lox) were bred with transgenic mice with cardiac-specific expression of Cre recombinase driven by the α-myosin heavy chain (α-MHC) promoter. Experiments were performed on mice with Con genotypes Cre:Glut4^+/+, not:Glut4^lox/lox, not:Glut4^lox/+, and not:Glut4^+/+ or loss of GLUT4 in cardiomyocytes (Cre:Glut4^lox/lox and G4H^−/−). No differences were noted between the different Con genotypes. Animals were fed standard chow (no. 8656, Teklad Diets, Madison, WI) and housed in temperature-controlled facilities with a 12:12-h light-dark cycle. All animal experiments were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee of the University of Utah.

Swimming exercise training.

Swimming exercise training was performed as previously described (17). Con and G4H^−/− mice (8–10 wk of age) underwent swim training 2 times/day, 7 days/wk, separated by at least 4 h (Swim groups). The swim duration was started from 10 min and increased by 10 min/day until mice were swimming 90 min. Mice were then trained at this duration for ~3 wk, and tissue was harvested for gravimetric and molecular measures.

TAC-induced pressure overload.

Aortic banding was performed as previously described (15, 29). Con and G4H^−/− mice (10–12 wk of age) were anesthetized and placed in the supine position on a heating pad (37°C). After a horizontal skin incision of ~1 cm in length at the level of the suprasternal notch, a ~3-mm longitudinal cut was made in the proximal portion of the sternum. TAC was implemented by placement of a metal clip calibrated to a 27-gauge-diameter needle between the innominate artery and left common carotid artery. The sham procedure was identical except that the aortic arch was not constricted. Mice were followed for ~3 wk, and tissue was harvested for gravimetric and molecular measures.

In vivo contractile function by echocardiography.

Mouse echocardiography was performed as previously described (28). Mice were anesthetized with isoflurane, and transthoracic two-dimensional guided M-mode images were recorded in both short- and long-axis projections using a linear 13-MHz probe on a Vivid FiVe device (GE Medical Systems, Milwaukee, WI). One operator, blinded to genotype and treatment, performed all echocardiograms.

Substrate metabolism in isolated working hearts.

Palmitate oxidation, myocardial O₂ consumption ($\mathrm{M}\dot{\mathrm{V}}{\mathrm{O}}_{\mathrm{2}}$) and ex vivo cardiac function were measured in isolated working hearts as previously described (21). Hearts were perfused with buffer containing [9,10-³H]palmitate with a final concentration of 0.4 mmol/l palmitate bound to 3% BSA and 5 mM glucose. Data were corrected to dry heart weight determined after perfusion.

Histological analysis.

Fresh hearts from three animals in each group were fixed by immersion in 10% buffered formalin and analyzed as previously described (8, 23). Tissue was embedded in paraffin, sectioned, and stained by manufacturer’s protocol with Masson’s trichrome or picrosirius red for visualization of fibrotic tissue. Apoptosis was determined by TUNEL (Roche, Basel, Switzerland) or by immunohistochemistry to measure cleaved caspase-3 (BD Biosciences, PharMingen, San Diego, CA). Additionally, an antibody to 4-hydroxy-2-nonenal (4-HNE; Oxis, Portland, OR) was used to detect lipid peroxidation. Light microscopy was performed using an Olympus IX71 inverted microscope (Olympus, Waltham, MA) equipped with a fluorescence filter at ×20 magnification.

Protein levels by Western blot analysis.

Total and phosphorylated proteins were measured by immunoblot analysis as previously described (8). Total protein lysates were prepared from frozen heart tissue (20–30 mg). Lysates (20–40 μg) were resolved by SDS-PAGE (8% or 10%) and electrotransferred onto polyvinylidene difluoride membranes (EMD Millipore, Darmstadt, Germany). Protein detection was performed with the appropriate horseradish peroxidase, IRDye 800, or Alexa Fluor 680 secondary antibodies, and chemiluminescence or fluorescence was quantified using film or LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE), respectively. Primary and secondary antibodies are shown in Table 1.

Table 1.

Antibody information for protein antigens examined by immunoblot analysis or immunohistochemistry

Antibody	Catalog No.	Company	Approximate Size, kDa	Source
Akt	05-591	EMD Millipore	60	Mouse
Phospho-Akt (Ser473)	9271	Cell Signaling Technology	60	Rabbit
Cleaved caspase 3	9661	Cell Signaling Technology	17, 19	Rabbit
4-HNE	24325	Oxis International	NA	Mouse
I2PP2A	sc-25564	Santa Cruz Biotechnology	39	Rabbit
PP2A C-subunit (α and β)	2259 2038	Cell Signaling Technology	36, 38	Rabbit
WGA-Alexa Fluor 488	W11261	Thermo Fisher Scientific	NA	NA
Anti-rabbit-Alexa Fluor 680	A10043	Invitrogen	NA	Donkey
Anti-rabbit-HRP	NA934	GE Healthcare	NA	Donkey
Anti-rabbit-IRDye 800	926-32213	LI-COR Biosciences	NA	Donkey
Anti-mouse-Alexa Fluor 680	A21058	Invitrogen	NA	Goat
Anti-mouse-IRDye 800	926-32212	LI-COR Biosciences	NA	Donkey

4-HNE, 4-hydroxynonenal; I2PP2A, inhibitor 2 of protein phosphatase 2A (PP2A); WGA, wheat germ agglutinin; HRP, horseradish peroxidase; NA, not applicable.

Ceramide and diacylglycerol levels.

Cardiac ceramide and diacylglycerol content were measured as previously described (14). Total lipids were extracted from cardiac tissue and quantified using a modified diacylglycerol assay kit.

Gene expression.

mRNA was quantified by real-time PCR as previously described (41). Total RNA was extracted from heart tissue with TRIzol reagent (Life Technologies) and purified with the RNeasy kit (Qiagen, Valencia, CA). Equal amounts of RNA were used to synthesize cDNA with Superscript III (Life Technologies). RT-PCR was performed using an ABI Prism 7900HT instrument (Applied Biosystems, Foster City, CA) in a 384-well plate format with SYBR green I chemistry and 6-carboxyl-X-rhodamine (ROX) internal reference (Life Technologies). Protein phosphatase 1A (Ppia) was used as a loading control. Primer sequences are shown in Table 2.

Table 2.

Gene target information and primer sequences used to quantify mRNA levels by quantitative PCR

Gene Symbol	Abbreviation	GenBank Accession No.	Gene Name	Forward Primer	Reverse Primer
Atp2a2	SERCA2A	NM_009722	ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2	5′-GAAACTACCTGGAACAACCCG-3′	5′-CTTTTCCCCAACCTCAGTCA-3′
Nppa	ANF	NM_008725	Natriuretic peptide precursor type A	5′-ATGGGCTCCTTCTCCATCA-3′	5′-CCTGCTTCCTCAGTCTGCTC-3′
Nppb	BNP	NM_008726	Natriuretic peptide precursor type B	5′-GGATCTCCTGAAGGTGCTGT-3′	5′-TTCTTTTGTGAGGCCTTGGT-3′
Myh6	α-MHC	NM_001164171	Myosin, heavy polypeptide 6, cardiac muscle, α	5′-ACTGTGGTGCCTCGTTCC-3′	5′-TTCCGTTTTCAGTTTCCGC-3′
Myh7	β-MHC	NM_080728	Myosin, heavy polypeptide 7, cardiac muscle, β	5′-CATTCTCCTGCTGTTTCCTTAC-3′	5′-CATGGCTGAGCCTTGGAT-3′
Ppargc1a	PGC-1α	NM_008904	Peroxisome proliferative activated receptor-γ coactivator-1α	5′-GTAAATCTGCGGGATGATGG-3′	5′-AGCAGGGTCAAAATCGTCTG-3′
Ppargc1b	PGC-1β	NM_133249	Peroxisome proliferator-activated receptor-γ coactivator-1β	5′-TGAGGTGTTCGGTGAGATTG-3′	5′-CCATAGCTCAGGTGGAAGGA-3′
Ppia	PPIA	NM_008907	Peptidylprolyl isomerase, also known as cyclophilin	5′-AGCACTGGAGAGAAAGGATTTGG-3′	5′-TCTTCTTGCTGGTCTTGCCATT-3′

Oxidative stress.

Tissue reactive oxygen species (ROS) levels were measured by the conversion of nonfluorescent 2′-7′-dichlorodihydrofluorescein diacetate (DCF-DA) to fluorescent 2′-7′-dichlorofluorescein in the presence of cellular esterases and endogenous ROS as previously described (42).

Mitochondrial enzyme activities.

Total carnitine palmitoyl-transferase (CPT), hydroxyacyl-coenzyme A dehydrogenase (HADH), and citrate synthase activity were measured in mitochondria as previously described (7).

Statistical analysis.

Data are expressed as means ± SE. An unpaired Student’s t-test was used to analyze data sets between two groups unless otherwise stated. Data sets with more than two groups were analyzed by two-way ANOVA to investigate main effects of genotype and treatment followed by Tukey’s multiple-comparison test when a significant interaction was observed; otherwise, Fisher’s least significant difference was used. For all analyses, P < 0.05 was accepted as indicating a significant difference. Statistical calculations were performed using Prism 7 (GraphPad Software, La Jolla, CA).

RESULTS

Cardiac hypertrophy is exacerbated after TAC in G4H^−/− mice.

To test whether enhanced glucose delivery by GLUT4 contributes to the cardiac adaptation to increased work after pressure overload, Con and G4H^−/− mice were subjected to TAC. The result was increased heart size in both genotypes (Fig. 1A). Consistent with an earlier report (2), nonstressed G4H^−/− hearts exhibited hypertrophy (Fig. 1A). Tibia length (TL) was unchanged between groups; thus, biventricular heart weight (BV) was corrected to TL. Relative to sham animals, BV/TL increased by ~16% in Con-TAC mice and by ~33% in G4H^−/−-TAC mice ~3 wk after surgery (Fig. 1B). Thus, BV/TL was similar in Con-TAC and G4H^−/−-sham animals, but G4H^−/−-TAC hearts were significantly more hypertrophied relative to all other groups (Fig. 1B). Evidence suggesting that this degree of hypertrophy was associated with elevated left ventricular (LV) filling pressures was provided by the finding of pulmonary edema in G4H^−/−-TAC mice (Fig. 1C). Cardiac tissue cellular remodeling was similar in the two TAC groups (Fig. 1D) with an equivalent increase in fibrosis in both TAC groups as examined by picrosirius red staining (Fig. 1E).

Fig. 1.

Loss of cardiac glucose transporter 4 (GLUT4) increases pathological remodeling after transverse aortic constriction (TAC). A: representative gross images of whole hearts from control (Con) and cardiomyocyte-specific GLUT4 knockout (G4H^−/−) mice ~3 wk after sham (Sham) or TAC surgery. B: biventricular heart weight (BV)-to-tibia length (TL) ratios. C: wet lung weight (WLW)-to-TL ratios. D: histological images of hematoxylin and eosin-stained cardiac sections (magnification: ×20). E: histological assessment of fibrosis by picrosirius red staining (magnification: ×20). Graphical data are shown as means ± SE; n = 9–15. *P < 0.05 vs. Con-Sham; $P < 0.05 vs. all. Two-way ANOVA showed a significant genotype effect on BV/TL and a significant treatment effect on BV/TL.

Accelerated decline in contractility in G4H^−/− mice after TAC.

Loss of GLUT4 accelerated progression to heart failure after TAC surgery, as determined by in vivo functional analysis by echocardiography and LV catheterization (Figs. 2 and 3). Heart rate was unchanged (Fig. 2B), but ejection fraction was significantly lower and LV internal diameter was significantly larger in the G4H^−/−-TAC mouse group versus all other groups (Fig. 2, A, C, and D). Posterior wall dimensions and interventricular septum thickness were unchanged (Fig. 2, E and F). The presence of LV dilation with normal wall thickness, rather than the expected geometric pattern of concentric hypertrophy with normal cavity size and increased wall thickness, may lead to a progressive cycle of increasing wall stress, further cavity enlargement, and worsening systolic function.

Fig. 2.

Echocardiographic analysis of in vivo cardiac dimensions and function in control (Con) and glucose transporter 4 knockout (G4H^−/−) mice ~3 wk after sham (Sham) or transverse aortic constriction (TAC) surgery. A: representative M-mode echocardiograms. B−F: heart rate (B), ejection fraction (C), and measures at diastole for left ventricular internal diameter (LVIDd), left ventricular posterior wall thickness (LVPWd), and interventricular septum thickness (IVSd; D–F). BPM, beats/min. Graphical data are shown as means ± SE; n = 9–15. $P < 0.05 vs. all. Two-way ANOVA showed a significant interaction of treatment and genotype for ejection fraction and LVIDd.

Fig. 3.

Left ventricular catheterization for in vivo hemodynamic measurements in control (Con) and glucose transporter 4 knockout (G4H^−/−) mice ~3 wk after sham (Sham) or transverse aortic constriction (TAC) surgery. A and B: left ventricular systolic pressure (LV SP) and end-diastolic pressure (LV EDP). C: rate of rise (+dP/dt) and fall (−dP/dt) of LV pressure. D: LV developed pressure (LV DevP). E: isovolumic relaxation constant (τ). Graphical data are shown as means ± SE; n = 5–7. *P < 0.05 vs. Con-Sham; #P < 0.05 vs. Con-TAC; ‡P < 0.05 vs. G4H^−/−-Sham. Two-way ANOVA showed a significant genotype effect on +dP/dt, −dP/dt, LV SP, and LV DevP and significant treatment effect on LV SP, LV EDP, LV DevP, and τ.

Blood pressure and contractility were evaluated in vivo by LV catheterization. LV systolic pressure after TAC in the G4H^−/− group was lower than the Con-TAC group, whereas LV end-diastolic pressure was comparable in the two groups (Fig. 3, A and B). Peak rates of LV pressure development (+dP/dt) and pressure decline (−dP/dt) were lowest in G4H^−/−-TAC mice (Fig. 3C). LV developed pressure was lower in G4H^−/−-TAC mice (Fig. 3D), and the isovolumic relaxation constant was prolonged in G4H^−/−-TAC mice (Fig. 3E). Therefore, loss of cardiac GLUT4 primes the heart for an accelerated decline in function when subjected to chronic pressure overload.

Exercise training did not alter heart weight but induced cardiac fibrosis in G4H^−/− mice.

We next sought to determine whether GLUT4-mediated glucose transport modulated the adaptations to the physiological stimulus of exercise training. Mice were subjected to a moderate training duration of 2- to 3-wk swim training and compared with sedentary (Sed) matched mice. After this duration of exercise training, BV corrected to body weight revealed a significant increase in both groups (Fig. 4A). However, as both Swim groups significantly lost body weight (Fig. 4B), we therefore compared BV-to-TL ratios, which were no longer increased (Fig. 4C). As such, this mild swim training was not sufficient to induce significant ventricular hypertrophy in Con animals, and although G4H^−/− had increased hypertrophy at baseline, there was no additional increase observed (Fig. 4C). To assess training intensity, skeletal muscle citrate synthase activity was measured in a subset of mice. There was a nonsignificant trend toward increased citrate synthase activity in Con-Swim mice, which contrasted with a significant twofold increase in G4H^−/−-Swim mice (Fig. 4D). This greater increase in G4H^−/− mice suggests a higher work level. We also examined cardiac histological samples for cellular morphology (Fig. 4, E and F) and fibrosis (Fig. 4, G and H). Cross-sectional quantification of cardiomyocyte size, as evidenced by wheat germ agglutinin cell area quantification (Fig. 4F), was comparable to the whole heart hypertrophy (Fig. 4C). Strikingly, we found evidence of fibrosis in G4H^−/−-Swim mice by either picrosirius red or trichrome staining (Fig. 4, G and H), suggesting pathological remodeling or relative ischemia.

Fig. 4.

Systemic and cardiac changes in control (Con) and glucose transporter 4 knockout (G4H^−/−) mice after ~3 wk of swim training (Swim). A−D: biventricular weight (BV)-to-body weight (BW) ratio (A), BW (B), BV-to-tibia length (TL) ratio (C), and gastrocnemius skeletal muscle whole cell lysate citrate synthase (CS) activity (D). E and F: histological examination using wheat germ agglutinin (WGA) with quantification. G and H: histological examination using picrosirius red and trichrome. Sed, sedentary. Graphical data are shown as means ± SE; n = 15–26 for A–C and n = 3–7 for D–H. *P < 0.05 vs. Con-Sed; #P < 0.05 vs. Con-Swim; ‡P < 0.05 vs. G4H^−/−-Sed. $P < 0.05 for two-way ANOVA genotype effect. There was a significant treatment effect on body weight, genotype effect on BV/TL, and interaction of treatment and genotype for CS activity.

GLUT4 deficiency modestly altered in vivo and ex vivo cardiac parameters after swim training.

Most echocardiographic parameters of LV function were not different between groups (Fig. 5). Although a few G4H^−/−-Swim mice had observable contractile dysfunction (Fig. 5A), the overall changes in ejection fraction trended down but did not reach statistical significance (Fig. 5C). LV internal diameter was greatest in G4H^−/−-Swim mice at diastole (Fig. 5D). Exercise training is associated with an increase in fatty acid utilization. We have previously reported a decrease in glucose uptake into G4H^−/− hearts (37). We therefore focused on ex vivo cardiac palmitate oxidation at baseline and in response to exercise. In Con hearts, exercise training was associated with a significant increase in FAO and $\mathrm{M}\dot{\mathrm{V}}{\mathrm{O}}_{\mathrm{2}}$. In contrast, although baseline FAO and MV̇o₂ in G4H^−/− mice were similar to those in Con mice, the exercise-induced increase in FAO and MV̇o₂ was prevented (Fig. 6, A and B). Of the ex vivo contractile measures, heart rate was higher in G4H^−/−-Swim mice (Fig. 6D), and LV diastolic pressure was significantly lowest in G4H^−/−-Swim mice (Fig. 6F). As fatty acid utilization was differentially regulated, we also measured cardiac diacylglycerol and ceramide levels in a separate cohort of mice and found that ceramide was decreased in Con-Swim mice but was unchanged in G4H^−/−-Swim mice (Fig. 6, H and I).

Fig. 5.

Echocardiography for in vivo cardiac measurements ~3 wk after swim training (Swim) or in sedentary (Sed) control (Con) and glucose transporter 4 knockout (G4H^−/−) mice. A−F: representative M-mode echocardiograms (A), heart rate (B), ejection fraction (C), and measures at diastole for left ventricular internal diameter (LVIDd), left ventricular posterior wall thickness (LVPWd), and interventricular septum thickness (IVSd; D–F). BPM, beats/min. Graphical data are shown as means ± SE; n = 12–18. *P < 0.05 vs. Con-Sed; #P < 0.05 vs. Con-Swim. Two-way ANOVA showed a significant genotype effect on LVIDd and a significant interaction of treatment and genotype for LVPWd.

Fig. 6.

Substrate oxidation and contractile function in isolated perfused working hearts (IWH). Palmitate oxidation (A), myocardial O₂ consumption (MV̇o₂; B), cardiac power (C), heart rate (D), cardiac output (E), left ventricular diastolic pressure (LVDP; F), and rate-pressure product (RPP; G) measured in IWH are shown. In a separate cohort of mice, cardiac tissue was snap frozen and used for diacylglycerol (H) or ceramide (I) measurements. Sed, sedentary; Swim, swim training; Con, control; dhw, dry heart weight; whw, wet heart weight; BPM, beats/min. Graphical data are shown as means ± SE; n = 6–11 for A–G and n = 5 for H and I. *P < 0.05 vs. Con-Sed; #P < 0.05 vs. Con-Swim. Two-way ANOVA showed a significant genotype effect on LVDP, a significant treatment effect on heart rate and cardiac output, and significant interaction of treatment and genotype for palmitate oxidation and MV̇o₂.

GLUT4 deficiency increased markers of cell death and altered cellular signaling after swim training.

Potential mechanisms for increased cardiac injury, apoptosis, and oxidative stress were evaluated in formalin-fixed hearts. Cleaved caspase-3 immunofluorescence (Fig. 7A) and increased TUNEL positivity (Fig. 7B) were only observed in G4H^−/−-Swim hearts. There were no significant changes in ROS as assessed by DCF-DA oxidation (data not shown), but a detectable increase in 4-HNE staining in G4H^−/− hearts independent of swim status was observed (Fig. 7C).

Fig. 7.

Histological examination of cardiac changes in control (Con) and glucose transporter 4 knockout (G4H^−/−) mouse hearts after ~3 wk of swim training (Swim). A−C: cleaved caspase-3 (A), TUNEL (B), and 4-hydroxynonenal (4-HNE; C). Sed, sedentary. Graphical data are shown as means ± SE; n = 3. *P < 0.05 vs. Con-Sed; #P < 0.05 vs. Con-Swim. Two-way ANOVA showed a significant genotype effect on 4-HNE.

To explore possible molecular mechanisms promoting increased cell death in exercise-trained G4H^−/− mice, we examined phosphorylation of the prosurvival kinase Akt. Similar to our prior study (17), Con-Swim mice exhibited increased Akt phosphorylation (Fig. 8A). However, despite increased basal levels of Akt phosphorylation, Akt was completely dephosphorylated after exercise training in G4H^−/− mice. The phosphorylation status of known upstream activators of Akt, such as 3-phosphoinositide-dependent protein kinase-1 and mechanistic target of rapamycin, was unchanged (data not shown). We therefore examined a negative regulator of Akt activity, the PP2A C-subunit (PP2A-C), which was modestly but significantly induced (Fig. 8A). We also examined the level of inhibitor 2 of PP2A (I2PP2A), which was modestly but significantly elevated in G4H^−/−-Sed mice but not in G4H^−/−-Swim mice (Fig. 8A).

Fig. 8.

Protein examination of cardiac changes in control (Con) and glucose transporter 4 knockout (G4H^−/−) mouse hearts after ~3 wk of swim training (Swim) and changes in gene expression and markers of mitochondrial oxidative capacity in nonstressed G4H^−/− mouse hearts. A: Western blot analysis of whole cell lysate from biventricular tissue. B: gene expression analysis by quantitative PCR (qPCR; see Table 2 for full gene names). C−E: carnitine palmitoyl-transferase (CPT) activity (C), hydroxyacyl-CoA dehydrogenase (HADH) activity (D), and citrate synthase activity (E). Sed, sedentary; a.u., arbitrary units. Graphical data are shown as means ± SE; n = 4–6 for A, n = 3 for B, and n = 4–6 for C–E. *P < 0.05 vs. Con-Sed; #P < 0.05 vs. Con-Swim; $P < 0.05 vs. G4H^−/−-Sed. Two-way ANOVA showed a significant genotype effect on the protein phosphatase 2A C-subunit (PP2A-C) and inhibitor 2 of PP2A (I2PP2A) and a significant interaction of treatment and genotype for phosphorylated (P-)Ser⁴⁷³ Akt.

Given the modest nature of these changes, we sought to determine whether additional but previously unrecognized molecular adaptations exist in G4H^−/− mouse hearts that could account for the maladaptive responses of these hearts. We observed a pattern of gene expression changes consistent with pathological remodeling that were already present before any stress (Fig. 8B). Specifically, there was a significant increase in transcript levels for atrial natriuretic factor, and there were significant decreases in transcript levels for α-MHC, SERCA2A, and peroxisome proliferator-activated receptor-γ coactivator-1β (PGC-1β), with a strong trend for PGC-1α (P = 0.12; Fig. 8B). In a separate cohort of mice, we also examined mitochondrial function for CPT, HADH, and citrate synthase activity (Fig. 8, C–E). There was a significant decrease in CPT activity (Fig. 8C), whereas HADH had a trend toward a decrease (P = 0.06), with relatively no change in citrate synthase activity. Taken together, these findings suggest that multiple mechanisms in GLUT4-deficient hearts increase their susceptibility to heart failure such as transcriptional profiles consistent with LV dysfunction at baseline, reduced mitochondrial fatty acid oxidative capacity, and impaired signaling to Akt.

DISCUSSION

In heart failure, cardiac metabolic substrate flexibility, utilization, and coupling to ATP production is reduced, which has been proposed as a contributing factor to the development of LV dysfunction (40). However, the role of substrate delivery under different conditions is not fully understood. Here, we provide evidence that GLUT4 is necessary for an adaptive cardiac response to enhanced hemodynamic demands by exercise or pressure overload. This finding supports the hypothesis that GLUT4, as opposed to GLUT1, is required for stress responses. These experiments further show that the requirement for GLUT4 also influences cell growth, survival, metabolism, and cellular signaling. The latter specifically appears to impact Akt signaling potentially through enhanced Akt dephosphorylation via PP2A. Recent studies have supported the hypothesis that an early enhancement of glucose uptake may precede the development of heart failure in response to pressure overload (18), with varying contributions from specific GLUT family members (34). However, our data suggest that in response to chronic deprivation of GLUT4, the responses to both pressure overload and exercise are maladaptive. Similarly, we have previously found that G4H^−/− mice with mild cardiac hypertrophy and preserved function were more susceptible to ischemic injury (37), despite higher glycogen content that was potentially fueled by increased GLUT1-mediated glucose uptake. However, the increase in GLUT1 was not sufficient to compensate for GLUT4 deficiency when hearts were subjected to hemodynamic stress (exercise or TAC). It is important to note that prior studies of G4H^−/− mice evaluated GLUT1 content only in nonstressed hearts (2). GLUT1 levels were not measured in the present study, and glucose uptake was not determined under the various conditions of hemodynamic stress that were induced. Therefore, to definitively test whether the impaired glucose uptake in G4H^−/− versus other potential changes in molecular signaling account for the changes observed, future studies will need to include glucose uptake and utilization measures postintervention. Early work describing the cardiac energy metabolic response to acute increases in workload supports an initial reliance on glycogen and lactate, switching to exogenous glucose by about an hour (12). Furthermore, hearts from G4H^−/− mice have elevated levels of atrial natriuretic peptide and brain natriuretic peptide (2), suggesting that they are primed toward pathological remodeling. The findings of the present study support the model that GLUT4 is the essential mediator of stress-mediated glucose utilization and highlight that an important function for GLUT4 may be its role in increasing glucose utilization to support increased cardiac work.

Differential roles of cardiac-enriched GLUTs.

A previous study by our group found that G1H^−/− mice did not exhibit accelerated progression to heart failure after TAC (26). In contrast, chronic lifelong GLUT1 overexpression in cardiomyocytes in vivo prevents adverse cardiac remodeling in response to pressure overload (19). Interestingly, when GLUT1 was expressed in the heart from birth, the elevated glucose delivery provided protection from ischemic injury in both young and old mice (20). However, when nutrient levels were increased by high-fat diet feeding, enhanced glucose delivery by GLUT1 overexpression increased oxidative stress and contractile dysfunction (43). Alternatively, short-term induction of GLUT1 in the adult mouse heart prevents mitochondrial dysfunction and attenuates structural remodeling but is not sufficient to prevent heart failure (25). Additionally, G4H^−/− mice exhibit increased levels of GLUT1 in the heart (2). Thus, the possibility exists that this increase in GLUT1 could provide cardioprotection. However, the present study reveals an obligate role for GLUT4-mediated glucose uptake in the cardiac adaptations to physiological or pathological hemodynamic stressors. Here, we show that loss of cardiomyocyte GLUT4 expression in the G4H^−/− mouse exacerbated the hypertrophic response to TAC and was associated with a more rapid decline in myocardial contractility. Moreover, cardiac muscle responds to exercise training by increasing FAO and mitochondrial capacity. Interestingly, loss of GLUT4 in G4H^−/− mouse hearts prevented the exercise-induced increase in myocardial FAO and MV̇o₂, suggesting that GLUT4-mediated glucose uptake might play an important role in the mitochondrial adaptations to exercise-induced cardiac hypertrophy via mechanisms that remain to be determined. One potential limitation of the present study was the use of swim training, as it has been suggested to be a nonphysiological stress (38). Future studies using other exercise models may provide insights into this.

Metabolic signaling to cell survival pathways.

Although these experiments support the need for additional glucose delivery in response to either chronic or intermittent stresses, we cannot rule out the contribution of other metabolic pathways. Many studies have focused on the balance of fatty acid and glucose utilization (1), but recent studies have suggested that the failing heart in humans and mouse models also uses ketones as an important fuel source (4, 5), supporting earlier observations (10, 32). A more comprehensive analysis of different metabolic substrates needs to be completed in the future to determine whether the cross talk between these pathways is similar to that proposed by Randle et al. (27) for fatty acid and glucose (35). In addition, it is now clear that metabolites play an active role in regulating protein function and gene expression (13, 39). One such example is the control of Akt signaling via ceramide-mediated regulation of PP2A-C, which has been demonstrated in the vasculature (6, 24, 45). It is also clear that signaling upstream of Akt (i.e., phosphatidylinositol 3-kinase) is essential for cardiac adaptations to physiological hypertrophy (22), consistent with the findings of the present study. A complete elucidation of the role of these mechanisms will require additional studies in this and other models of GLUT gain and loss of function.

Additional potential mechanisms contributing to the maladaptive phenotype of GLUT4-deficient hearts.

Additional mechanisms likely contribute to the dysfunction unveiled after hemodynamic stress in G4H^−/− hearts. Although not directly assessed in the present study, it is likely that the energy-sensing pathway of AMP-activated protein kinase-α or the oxygen-sensing pathway of hypoxia-inducible factor-1α, which both play critical roles in the cardiac adaptation to stress, could be altered in G4H^−/− hearts and contribute to their maladaptive responses (11, 18, 33, 44), and this warrants future study. An interesting new finding in the present study is that the otherwise mild hypertrophy seen in G4H^−/− mice is associated with gene expression changes and altered mitochondrial metabolism that is consistent with pathological LV remodeling. We therefore propose that these changes in concert with the heart’s inability to increase glucose uptake act synergistically to promote a maladaptive response even to a mild exercise stimulus.

Technical limitations of in vivo measures of cardiac structure and function.

Some technical limitations in the measures of functional and cardiac remodeling warrant discussion. Specifically, an important limitation of the echocardiography data collected was the absence of wall thickening (Fig. 2) in Con mice 3 wk after TAC despite elevated gravimetric evidence of hypertrophy (Fig. 1). Similar technical limitations of the measurements of cardiac function were also noted in the LV catheterization data collected in the TAC experiments, such as the absence of a significant difference between G4H^−/− and Con TAC mice in LV end-diastolic pressure (Fig. 3). Although these limitations do not alter the core conclusions of this study, additional and more sensitive measures of functional parameters may be needed in future studies.

Conclusions.

Taken together, this study and the published literature support the hypothesis that cardiomyocyte GLUT4 expression plays an important role in the cardiac adaptation to increased hemodynamic demands. In contrast to GLUT1, when GLUT4 is deleted from the heart, cardiac hypertrophy is exacerbated, and there is an accelerated decline in contractile function in response to pressure overload. Interestingly, an important requirement for GLUT4 is also seen in the cardiac response to exercise training. In the case of increased demands of exercise, G4H^−/− mice exhibited increased cell loss and myocardial fibrosis that correlated with a reduced capacity to oxidize fatty acids and inactivation of Akt. It is important to note that some of these maladaptive responses could result from additional underlying molecular changes (e.g., reduced PGC-1α). Furthermore, it is also important to note that the present study does not represent an exhaustive examination of all potential mechanisms linked to the development of heart failure. Long-term loss of GLUT4 induces pathological LV remodeling that is not ameliorated by exercise but rather sensitizes the heart to additional contractile dysfunction. Thus, although GLUT4 might be dispensable in the nonstressed heart, it serves an obligate role in the adaptation to hemodynamic stress.

GRANTS

A. R. Wende was supported by American Heart Association (AHA) Grant 725064Y and National Heart, Lung, and Blood Institute Grant 111322. E. D. Abel, an Established Investigator of the AHA, was also supported by National Institutes of Health Grants R01-DK-092065 and U01-HL-087947.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.R.W. and E.D.A. conceived and designed research; A.R.W., J.K., W.L.H., B.E.W., B.T.O., J.T., M.K.B., M.E.P., M.A.M., I.L., and G.V.H. performed experiments; A.R.W., J.K., W.L.H., B.E.W., B.T.O., J.T., M.K.B., M.E.P., M.A.M., I.L., G.V.H., S.E.L., and E.D.A. analyzed data; A.R.W., B.T.O., and E.D.A. interpreted results of experiments; A.R.W. prepared figures; A.R.W. and E.D.A. drafted manuscript; A.R.W., J.K., W.L.H., B.E.W., B.T.O., J.T., M.K.B., S.E.L., and E.D.A. edited and revised manuscript; A.R.W., J.K., W.L.H., B.E.W., B.T.O., J.T., M.K.B., M.E.P., M.A.M., I.L., G.V.H., S.E.L., and E.D.A. approved final version of manuscript.