Loss of the eIF2α kinase GCN2 protects mice from pressure overload induced congestive heart failure without affecting ventricular hypertrophy

Abstract

In response to a number of stresses, including nutrient deprivation, General Control Nonderepressible 2 kinase (GCN2) attenuates mRNA translation by phosphorylating eukaryotic initiation factor 2 alpha (eIF2α^Ser51). Energy starvation is known to exacerbate congestive heart failure (CHF) and eIF2α^Ser51 phosphorylation is increased in the failing heart. However, the impact of GCN2 during the evolution of CHF has not been tested. In this study we examined the influence of GCN2 expression in response to a cardiac stress by inducing chronic pressure overload with Transverse Aortic Constriction (TAC) in Wild Type (WT) and GCN2 knockout (GCN2^−/−) mice. Under basal conditions, GCN2^−/− had normal LV structure or function but following TAC, demonstrated less contractile dysfunction, less increase of lung weight, less increase of lung inflammation and vascular remodeling, and less myocardial apoptosis and fibrosis compared with WT mice, despite an equivalent degree of LV hypertrophy. As expected, GCN2^−/− attenuated TAC induced cardiac eif2α^Ser51 phosphorylation and preserved Sarcoplasmic reticulum Ca²⁺ ATPase (Serca2a) expression compared with WT mice. Interestingly, expression of the anti-apoptotic protein Bcl-2 was significantly elevated in GCN2^−/− hearts, while in isolated neonatal cardiomyocytes, selective knockdown of GCN2 increased Bcl-2 protein expression and enhanced myocyte resistance to an apoptotic stress. Collectively, our data support the notion that GCN2 impairs the ventricular adaptation to chronic pressure overload by reducing Bcl-2 expression and increasing cardiomyocyte susceptibility to apoptotic stimuli. Our findings suggest that strategies to reduce GCN2 activity in cardiac tissue may be a novel approach to attenuate congestive heart failure development.

Introduction

Congestive heart failure (CHF) is a major cardiovascular disease of epidemic proportion that has increased in prevalence in the past few decades. Several recent studies have indicated that amino acids or branched-chain amino acids (BCAA) may play an important role in maintaining cardiac function during normal conditions¹ and following the development of congestive heart failure^{2, 3}. Not surprisingly, BCAA dietary supplements promote cardiac and skeletal muscle mitochondrial biogenesis and prolong survival in mice¹, while chronic oral amino acid supplements improve cardiac function and exercise capacity in the elderly patient, both with normal and abnormal cardiac function^{2, 3}. Consistent with the important role of amino acid metabolism in cardiac function, Wang and colleagues demonstrated in a landmark study that protein phosphatase 2Cm (PP2Cm), a critical enzyme for BCAA metabolism, is dramatically reduced in the failing hearts, and that disrupting BCAA metabolism by inactivation of PP2Cm caused a significant decrease in cardiac contractility with premature death in zebra fish. These data highlighted the important role of BCAA metabolism in cardiac development and normal cardiac function⁴.

Amino acid availability is sensed by eIF2α kinase General Control Nonderepressible 2 kinase (GCN2). Amino acid deprivation, particular BCAA deprivation, increases the levels of uncharged tRNAs which bind the histadyl-tRNA synthethase-like domain of GCN2, resulting in its activation⁵. Upon activation, GCN2 phosphorylates eukaryotic initiation factor 2 alpha (eIF2α^Ser51), which attenuates global mRNA translation, while inducing a selective subset of stress response genes, including ATF4 and CHOP⁶. Conversely, when levels of amino acids or BCAA are sufficient to support translation, uncharged intracellular tRNAs are less abundant, and GCN2 activity is reduced. While studies have clearly demonstrated that GCN2 plays a critical role for amino acid sensing and the translation initiation process, the effect of amino acid sensor GCN2 on pressure overload induced CHF is unrecognized.

In this study, we tested the impact of the eIF2α kinase GCN2 pathway during development of CHF by inducing chronic pressure overload with TAC in mice with genetic disruption of the GCN2 gene (GCN2^−/−) and compared the results with WT mice. Although GCN2^−/− did not alter cardiac structure and function under basal conditions, it significantly attenuated TAC-induced heart failure, as evidenced by decreased ventricular dysfunction and dilatation as well as reduced pulmonary congestion. Of interest, GCN2^−/− increased the expression of the anti-apoptotic factor Bcl-2, while reducing both cardiac oxidative stress and cardiomyocyte apoptosis. Taken together, these data indicate that inhibition of GCN2 activity attenuates CHF development.

Methods

An extended Material and Methods section can be found in the online-only Data Supplement.

Mice and TAC

Male C57BL/6 (Taconic, Germantown, NY) and GCN2^−/− mice⁷ (congenic with the Taconic C57BL/6 strain), 8–10 weeks of age, were used. This study was approved by the Animal Care and Use Committee of the University of Minnesota. The TAC procedure was performed on wild type (n=15) and GCN2^−/− mice (n=17) as previously described^{8, 9}. Body weight and age matched wild type mice (n=7) and GCN2^−/− mice (n=8) were used as controls.

Echocardiography and evaluation of LV hemodynamics

Mice were anesthetized with 1.5% isoflurane. Echocardiographic images were obtained with Veve 660 system as previously described^{8, 9}.

Western blots

Protein content was analyzed using Western blots. The sources of the primary antibodies are listed in Table S1.

Quantitative Real-time PCR

Total RNA was reverse-transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase (Clontech). The real-time PCR reaction was carried out using a Light Cycler Thermocycler (Roche Diagnostics). Primers are listed in Table S2. Results were normalized to 18S rRNA.

Data and Statistical Analysis

All values are expressed as mean ± standard error. Statistical significance was defined as P < 0.05. One-way analysis of variance (ANOVA) was used to test each variable for differences among the treatment groups with StatView (SAS Institute Inc). If ANOVA demonstrated a significant effect, pair wise post hoc comparisons were made with Fisher’s least significant difference test.

Results

GCN2^−/− had no effect on LV structure and function under control conditions and TAC-induced LV hypertrophy

LV structure and function were not different between GCN2^−/− and WT mice under control conditions, as indicated by similar LV weight, lung weight, LV fibrosis and LV ejection fraction in these mice (Figure 1A–D, Figure S1). Exposure to TAC for 4 weeks also caused similar increases of LV weight and its ratio to body weight or tibia length in wild type and GCN2^−/− mice (5.90±0.29 in WT mice vs 6.04±0.27 in GCN2^−/− mice) (Figure 1A, Table S3), indicating that GCN2^−/− has no effect on TAC-induced LV hypertrophy.

Figure 1. Effects of GCN2 KO on TAC induced hypertrophy, pulmonary congestion and LV dysfunction.

After 4 weeks of TAC or control conditions, tissue was collected from WT and GCN2 KO mice and used to measure left ventricular weight (A) and lung weight (B). LV fibrosis was analyzed using Sirius red staining (C). Echocardiography was used to measure LV ejection fraction (D), LV end systolic diameters (E), end diastolic diameters (F), heart rates (G), and LV wall thickness (H) in WT and GCN2 KO mice. * indicates p<.05 comparing TAC to control. # indicates p<.05 comparing WT to GCN2 KO.

GCN2^−/− attenuated TAC-induced LV dysfunction

Despite similar levels of cardiac hypertrophy, the increases in lung weight and its ratio to body weight (10.25±1.35 in wild type mice as compared with 7.14±0.92 in GCN2^−/− mice) or tibia length were significantly higher in WT mice exposed to TAC than in GCN2^−/− mice (Figure 1B, Table S3). These data suggest that genetic deletion of GCN2 reduces TAC-induced pulmonary congestion, a highly reliable marker for LV dysfunction¹⁰. Echocardigraphy further confirmed that GCN2^−/− mice developed significantly less LV dysfunction in response to TAC as indicated by significantly less reduction in LV ejection fraction (56±3.7% in WT vs 69±3.8% in GCN2^−/−; p<0.05) and significantly less increases of LV end systolic diameter (3.30±0.18mm in WT vs 2.75±0.21mm in GCN2^−/−; p<0.05) and LV end diastolic diameter (4.37±0.13mm in WT vs 4.08±0.15mm in GCN2^−/−; p<0.05) (Figure 1D–F). TAC caused a small but significant reduction of heart rate in both WT and GCN2^−/− mice (Figure 1G). Both WT and GCN2^−/− mice developed significant but similar increases in LV wall thickness at the end diastole (Figure 1H).

Despite the similar amount of LV hypertrophy in wild type and GCN2^−/− hearts, histological staining revealed that GCN2^−/− hearts develop significantly less LV fibrosis than WT hearts exposed to TAC (12.2±0.64% in WT vs 7.85±0.61% in GCN2^−/−) (Figure 1C, Figure S1). In addition, GCN2^−/− hearts exhibited significantly less Leukocyte accumulation than WT hearts exposed to TAC (leukocytes are revealed by CD45 positive staining) (Figure S2). TAC also increased collagen-I mRNA expression more strongly in WT hearts than in GCN2^−/− hearts (1.28±0.1299 in WT vs 0.76±0.17 in GCN2^−/−) (Figure S3). These findings clearly indicate significantly better cardiac adaptation in GCN2^−/− hearts after TAC.

GCN2^−/− attenuated heart failure induced lung vascualr remodeling and lung inflammation

We previously reported that LV dysfunction causes significant lung remodeling¹⁰, which is anticipated to exacerbate the progression of heart failure. Therefore, we further determined the TAC-induced lung vascular remodeling and inflammation in WT and GCN2^−/− mice (Figure 2). The result demonstrated that the percentage of non-muscularized (NM), partially muscularized (PM), and fully muscularized small arteries (FM) in lung tissues were not different between wild type and GCN2^−/− mice under control conditions (Figure 2A,D). TAC caused increases in fully muscularized small arteries in both wild type and GCN2^−/− mice, but these increases were greater in the wild type mice than in GCN2^−/− mice (Figure 2D). Both wild type and GCN2^−/− mice had significant decreases in non-muscularized small arteries but these decreases were significantly greater in wild type mice than in GCN2^−/− mice (Figure 2D), indicating that GCN2^−/− significantly attenuated heart failure induced lung vascular remodeling. In addition, GCN2^−/− dramatically attenuated TAC-induced lung inflammation as indicated by less accumulation of leukocytes and macrophages in the alveolae, in the vascular wall of large vessels and in the interstitial space of lung tissues (Figure 2B–C). The increased infiltration of macrophages was confirmed by staining with the macrophage-specific marker Mac-2 (Figure 2B), while the infiltration of neutrophils was confirmed using a specific antibody against mouse neutrophil clone 7/4 (Figure 2C). Neither GCN2^−/− nor TAC affected the relative lung water content (Figure S4), a finding that is consistent with our previous report that the relative lung water contents were not changed in CHF animals¹⁰.

Figure 2. GCN2 influences TAC induced lung vascular remodeling and lung inflammation.

After 4 weeks of TAC or control conditions, tissue sections were stained with hematoxylin and eosin (A), macrophage-specific marker Mac-2 (B, brown staining) and antibody specific for neutrophil clone 7/4 (C, brown staining). The distribution of nonmuscular, partially muscular, and fully muscularized small arteries in WT and GCN2 KO mice under control conditions and after TAC were summarized (D)* indicates p<.05 comparing TAC to control. # indicates p<.05 comparing WT to GCN2 KO.

GCN2^−/− attenuated TAC-induced phosphorylation of eIF2α

eIF2α is the only substrate of GCN2 that has been identified. However, the specific contribution of GCN2 to eIF2α^Ser51 phosphorylation in the heart under basal or stress conditions is not known. Western blot analysis demonstrated that both phosphorylated GCN2^Thr667 and total GCN2 are expressed in the heart, and completely absent in GCN2^−/− hearts, as expected (Figure S5A–D). The phosphorylated GCN2^Thr667 was significantly increased in WT hearts after TAC. The absence of GCN2 protein did not significantly influence total LV eIF2α content or eIF2α^Ser51 phosphorylation under basal conditions. However, while eIF2α^Ser51 phosphorylation was increased in WT hearts in response to TAC, this increase was blunted in GCN2^−/− hearts (Figure S5A, E–G). These data suggest GCN2 plays little role in cardiac eIF2α^Ser51 phosphorylation under basal conditions (or that its loss is compensated for by other eIF2α kinases), but does contribute to increased phosphorylation of eIF2α^Ser51 under the stress conditions imposed by systolic overload.

GCN2^−/− attenuated TAC-induced decrease of Serca2a and increase of autophagy, oxidative stress and ER Stress

Western blot analysis of LV lysates demonstrated that GCN2^−/− blunted the increase of atrial natrurietic peptide (ANP; a marker for cardiac stress) and prevented the loss of Sarcoplasmic reticulum Ca²⁺ ATPase (Serca2a), an enzyme that is essential for normal cardiac function. Since CHF is associated with increased ER stress¹¹, autophagy¹² and oxidative stress, markers for cardiac ER stress, autophagy and oxidative stress were determined. ER stress marker CHOP is undetectable in WT and GCN2^−/− hearts under control conditions, while TAC caused a significant increase of CHOP expression in WT and GCN2^−/− hearts. GCN2^−/− signficantly attenuated myocardial CHOP expression in mice after TAC (Figure S6A,B). GCN2^−/− also reduced the TAC induced up-regulation of ER chaperone GRP78 (Figure S6A,C), while the TAC-induced increase of GRP94 also tended to be less in the GCN2^−/− mice (p=0.10) (Figure S6A,D). Autophagy marker beclin-1 was significantly increased in GCN2^−/− hearts under control conditions, while autophagy marker LC-3B was unchanged in GCN2^−/− hearts under control conditions (Figure 3A,D,E). TAC caused significant increases of beclin-1 and LC-3B in WT hearts, while the increases of these proteins were significantly attenuated in GCN2^−/− hearts (Figure 3A, D, E).

Figure 3. GCN2 effects on TAC induced changes in ANP, Serca2a, autophagy markers and oxidative stress.

Tissue was collected from WT and GCN2 KO mice 4 weeks after TAC or control conditions, and lysates were examined by western blot for expression of atrial natrurietic peptide (ANP), Serca2a, autophagy makers Beclin-1 and LC-3B, and oxidative makers 3′-NT and 4-HNE. Sarcomeric α-actin was used as a loading control. * indicates p<.05 comparing TAC to control. # indicates p<.05 comparing WT to GCN2 KO.

Since both the increase of Serca2a expression and the decrease of CHOP expression attenuate pressure overload induced myocardial oxidative stress and CHF^{13, 14}, we speculated that GCN2^−/− might attenuate myocardial oxidative stress after TAC. Consequently, myocardial oxidative stress markers 3′-Nitrotyrosine and 4-HNE were determined by western blot. Myocardial 3′-Nitrotyrosine (3-NT) and 4-HNE contents were not different in WT and GCN2^−/− hearts under control conditions. However, whereas TAC caused significant increases of 3-NT and 4-HNE in WT hearts, the increase in oxidative markers was significantly attenuated in GCN2^−/− hearts (Figure 3A, F, G). The reduced ANP expression, preserved Serca2a expression and reduced oxidative stress in GCN2^−/− mice exposed to TAC indicate GCN2 plays a pathological role in the response to hemodynamic overload.

GCN2^−/− increased LV Bcl-2 expression and attenuated TAC-induced apoptosis

Cardiomyocyte death and replacement fibrosis are features of pathological hypertrophy that cause LV dysfunction. To determine if GCN2 affects cardiomyocyte apoptosis during pressure overload, Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed on hearts from WT and GCN2^−/− mice exposed to TAC. While TAC significantly increased apoptotic cells both in WT and GCN2^−/− hearts, the increase in apoptotic cells was significantly less in GCN2^−/− hearts than in WT hearts (Figure 4A, Figure S7). Consistent with TUNEL staining results, WT hearts exhibited greater activation (cleavage) of pro-apoptotic protein caspase-3 in response to TAC than GCN2^−/− hearts (Figure 4B,C). Caspase-3 cleavage occurs downstream of mitochondrial cytochrome-C release, which is promoted by the pro-apoptotic factor Bax, and inhibited by the anti-apoptotic factor Bcl-2. The ratio of Bcl2 to Bax is thus an important indicator of apoptotic sensitivity. To investigate potential mechanism(s) by which GCN2 might increase sensitivity to TAC induced apoptosis, we examined expression of Bcl-2 and Bax in WT and GCN2^−/− hearts. We observed that Bax expression was similar in WT and GCN2^−/− hearts under basal conditions. Bcl-2 expression, however, was significantly elevated in GCN2^−/− hearts even under basal conditions, indicating that GCN2 limits steady state Bcl-2 levels in the heart. In response to TAC, Bax levels increased significantly in WT hearts, but this increase was blunted in GCN2^−/− hearts, suggesting GCN2 contributes to increased Bax expression in pressure overload (Figure 4B,D). Because increased Bcl-2 expression was maintained in GCN2^−/− hearts after TAC (Figure 4B,E), while the increase in Bax observed in WT hearts was blunted by GCN KO, the ratio of Bax/Bcl-2 was significantly higher in WT hearts after TAC as compared to GCN2^−/− hearts(Figure 4F). We also measured the Bcl-2 mRNA level in those experimental groups. It was found that Bcl-2 mRNA was significantly increased in GCN2^−/− hearts under control conditions but not after TAC, suggesting that the increase of Bcl-2 protein expression in GCN2^−/− hearts is regulated at both transcriptional and post-transcriptional levels (Figure S8). These data suggest GCN2 may increase cardiomyocyte susceptibility to apoptosis during pressure overload by down-regulating Bcl-2 expression and elevating expression of Bax.

Figure 4. GCN2 effects on TAC induced cardiac cell apoptosis.

After 4 weeks exposure to TAC or control conditions, LV tissue from WT and GCN2 KO mice was fixed and analyzed for apoptotic events using TUNEL staining (A). LV lysates were also examined by western blot for caspase-3 cleavage (B, C), Bcl-2 expression (B, D), and Bax expression (B, E). The Bax/Bcl-2 ratio was also listed (F). * indicates p<.05 comparing TAC to control. # indicates p<.05 comparing WT to GCN2 KO.

GCN2 regulates Bcl2 expression and apoptosis in cultured cardiac myocytes

The observation that Bcl2 expression was significantly increased in LV tissue obtained from GCN2^−/− mice, and that GCN2^−/− hearts exhibited less TAC induced apoptosis than WT mice, suggests a novel role for GCN2 in cardiomyocyte apoptotic sensitivity, in part through regulation of Bcl-2. However, because GCN2 is disrupted in the germ-line of these mice, the Bcl-2 increase and protection against apoptosis in the heart could result from an adaptive embryonic response to loss of GCN2 or from non-cardiomyocyte effects. Therefore, we used an isolated neonatal rat cardiac myocyte model to further investigate the relationship between GCN2 and Bcl-2 expression and examine how this influences cardiomyocyte apoptosis. In neonatal cardiomyocytes, GCN2 was expressed at low levels under basal conditions, but was significantly increased by extended hypertrophic stress (50 μM phenylephrine for 72 hours) (Figure 5A, B). Interestingly, increased GCN2 expression was associated with reduction of Bcl-2 expression in response to phenylephrine treatment (Figure 5A, C). To determine if GCN2 expression contributes to Bcl-2 reduction in hypertrophied cardiomyocytes, we depleted GCN2 levels using RNAi. Consistent with our findings from mice studies, treatment of cardiomyocytes with GCN2 targeting RNAi effectively reduced GCN2 expression and significantly increased Bcl-2 expression in comparison to non-targeting RNAi treated controls (Figure 5A–C). In addition, Bcl-2 expression was better preserved in GCN2 RNAi treated cardiomyocytes than controls after exposure to extended phenylephrine treatment (72 hours). While TUNEL staining did not reveal significant differences in apoptosis between control and GCN2 depleted cells under basal conditions or after phenylephrine treatment, GCN2 depleted cells were more resistant to apoptosis induced by additional exposure to an oxidative stress from H₂O₂ (Figure 5D). Conversely, we found that overexpression of GCN2 mouse active kinase domain significantly attenuated Bcl-2 expression in neonatal cardiomyocytes after PE treatment (50μM, 72h). GCN2 overexpression also exacerbated apoptosis induced by additional exposure to oxidative stress from H₂O₂ (Figure S9). Collectively, these results suggest that GCN2 attenuates Bcl2 expression in cardiomyocytes and increases susceptibility of cardiomyocytes to an apoptotic stress.

Figure 5. RNAi depletion of GCN2 and the influence on Bcl-2 expression and apoptosis during hypertrophic stress in neonatal rat cardiomyocytes.

Neonatal rat cardiomyoctes were transfected with RNAi targeting GCN2 or non-targeting control RNAi overnight, and then cultured under basal conditions or treated with 50μM phenylephrine (PE) for an additional 72 hours. Protein lysates were examined by western blot (n=5 to 6 samples) for expression of GCN2 (A, B) or Bcl-2 (A, C). Some wells were treated with 100μM H₂O₂ for the last 24 hours, followed by TUNEL staining to measure apoptosis (D). * indicates p<.05 comparing TAC to control. # indicates p<.05 comparing WT to GCN2 KO.

Discussion

The present study provides the first direct evidence that inhibition of GCN2 activity by GCN2 gene deletion attenuates the development of heart failure. GCN2 deletion did not reduce the degree of cardiac hypertrophy in response to chronic pressure-overload, but did attenuate the degree of ventricular dysfunction and dilatation as well as the amount of pulmonary congestion. Additional evidence for a pathological role for GCN2 in response to a cardiac stress was reduced ANP content, preserved Serca2a expression, and attenuated ER stress response observed in GCN2^−/− hearts after TAC. The finding that GCN2^−/− protects against heart failure during chronic pressure overload indicates that selective inhibition of GCN2 may be a therapeutic target for treating heart failure.

The finding that GCN2 deletion increases Bcl-2 expression and protects against cardiomyocyte apoptosis suggests a potential mechanism for the maladaptive effects of GCN2 in pressure overload. Cardiomyocyte apoptosis is believed to contribute to the transition from compensatory hypertrophy to heart failure¹⁵. Bcl-2 is a well-recognized anti-apoptotic factor that protects against oxidative stress-induced apoptosis¹⁶ and can preserve cardiomyocyte viability and LV function under a variety of stress conditions when over-expressed in the heart^17–19. The finding that GCN2^−/− mice exhibit increased Bcl-2 expression and decreased cardiac cell apoptosis identifies GCN2 as a novel negative regulator of cardiac Bcl-2 expression. GCN2 regulation of cardiac Bcl-2 and apoptosis was further confirmed in an isolated neonatal cardiomyocyte hypertrophy model, where phenylephrine increased GCN2 expression and reduced Bcl-2 expression, while RNAi depletion of GCN2 increased Bcl-2 expression and improved resistance to hydrogen peroxide induced apoptosis. Interestingly, while GCN2 limits cardiac Bcl-2 expression, GCN2 increases cardiac expression of the pro-apoptotic factor Bax during systolic overload. These pro and anti apoptotic factors interact with each other and have opposing effects on mitochondrial cytochrome c release²⁰, a critical step in caspase activation and apoptosis. Accordingly, the reduction of Bcl-2 and increase of Bax expression by GCN2 is likely to increase cardiomyocyte susceptibility to TAC induced apoptosis^{20, 21}.

While our data identify a novel role for GCN2 in Bcl-2 expression and apoptosis, the mechanism by which GCN2 regulates Bcl-2 levels is not totally clear. Based on previous findings, the increased ER stress response observed in WT mice (in comparison to GCN2^−/− mice) may lower Bcl-2 expression by increasing expression of CHOP^{13, 22}. CHOP has been demonstrated to reduce transcription of Bcl-2²², thereby increasing apoptosis in response to ER stress²³. Recently CHOP was also shown to contribute to cardiomyocyte apoptosis and LV dysfunction in pressure overload¹³. Thus GCN2 mediated increase in CHOP under stress conditions may contribute to the reduced Bcl-2. However, while attenuated phospho-eif2α/CHOP activity may help explain how GCN2 ablation preserves Bcl-2 expression under stress conditions, we observed that Bcl-2 expression is also elevated in GCN2^−/− hearts under basal conditions, during which no difference is observed between WT and GCN2^−/− hearts in eif2α phosphorylation. Similarly, in isolated cardiomyocytes, we did not observe large differences in eif2α phosphorylation in GCN2 depleted cells, even as Bcl-2 expression was increased. These finding suggest that either a moderate change of eif2α phosphorylation is sufficient to regulate Bcl-2 expression, or GCN2 regulates Bcl-2 levels independent of eif2α phosphorylation.

Serca2a is a well recognized therapeutic target for treating congestive heart failure²⁴. Previous studies have consistently demonstrated that decreased myocardial Serca2a exacerbates CHF development²⁵, while increased Serca2a expression attenuates CHF development²⁴. In that regard, the reduced Serca2a reduction in the GCN2^−/− mice after TAC provides an alternate important mechanism for improved cardiac function. It has been consistently demonstrated that increased myocardial oxidative stress exacerbates CHF in response to chronic pressure overload, while decreased myocardial oxidative stress attenuates CHF^{8, 13}. The reduced myocardial CHOP expression and lower levels of oxidative stress observed in the GCN2^−/− mice after TAC may also contribute to the improved cardiac function in these mice. The finding that CHOP expression was reduced in GCN2^−/− hearts after TAC is consistent with the previous report that CHOP induction under stress conditions is partially GCN2 dependent⁷. Several studies showed that CHOP expression is increased in the failing heart^{13, 26}, while CHOP gene deletion attenuates TAC- induced myocardial oxidative stress and heart failure¹³ and oxidative stress in beta cells in multiple mouse models of diabetes²⁷. Therefore, the reduced CHOP expression observed in GCN2^−/− mice after TAC may contribute to reduced myocardial oxidative stress.

Amino acid deprivation or nutrient starvation activates GCN2 activity, while amino acid administration (particularly administration of BCAA) attenuates GCN2 activity. However, studies have also demonstrated that GCN2 can be activated by other stresses such as glucose deprivation²⁸, infection²⁹, oxidative stress³⁰ and UV radiation³¹. The dramatic changes in myocardial metabolism and other mechanical and biochemical stresses in the failing heart may all contribute to GCN2 activation directly or indirectly. The increased phosphorylation of eIF2α and GCN2 in wild type mice after TAC suggests an increased activity of GCN2 in the failing hearts, but the mechanism of GCN2 activation in the failing heart is not clear. A recent study demonstrated that chronic administration of high dose BCAA prolonged survival in mice. The prolonged survival due to BCAA treatment was associated with increased expression of genes involved in mitochondrial biogenesis, antioxidant defenses and marked reduction of ROS production in cardiac and skeletal muscles of wild type mice but not eNOS KO mice¹. Inhibition GCN2 activity through GCN2 gene deletion did not result in increased expression of genes involved in mitochondrial biogenesis and antioxidant defense in skeletal muscles in mice, at least under control conditions^{13, 26}, suggesting different molecular pathways are involved in the above conditions^{13, 26}.

The present study has several limitations. First, GCN2 gene deletion significantly increased myocardial Bcl-2 expression, attenuated TAC-induced Serca2a expression, autophagy, ER stress and oxidative stress. The cardiac protective effect observed in GCN2^−/− mice is likely to be a collective effect of all of these changes. We are unable to determine the relative contribution of these individual factors for the cardiac protective effect in this mouse strain. In addition, as eif2α plays an essential role for translation of mRNAs into proteins, while GCN2^−/− only moderately attenuated TAC-induced LV dysfunction, it is difficult to determine whether eif2α is required for GCN2 effects on Bcl-2 expression in the current experimental setting. In addition, eif2α plays an essential role for translation of mRNAs into proteins, so disruption of eif2α is anticipated to exert effects on cardiomyocyte survival far beyond GCN2. Therefore, we are unable to determine whether eif2α is required for GCN2 effects on Bcl-2 expression in the current experimental setting. Finally, as a global GCN2^−/− mouse strain was used for the present study, some of the observed phenotypes may be a result of chronic adaptation to the global gene deletion. Moreover, the present study could not determine whether the cardiac protective effect is due to the gene deletion in cardiomyocytes or gene deletion in other cell types.

Perspectives

Amino acid availability is sensed by eIF2α kinase GCN2. While studies have clearly demonstrated that GCN2 plays a critical role for amino acid sensing and the translation initiation process, the effect of amino acid sensor GCN2 on heart failure development is unrecognized. Our study provides the first direct evidence that inhibition of GCN2 activity by GCN2 gene deletion attenuates the development of heart failure and the amount of pulmonary remodeling. The finding that GCN2^−/− protects against heart failure during chronic pressure overload indicates that selective inhibition of GCN2 may be a therapeutic target for treating heart failure.

Novelty and Significance.

What Is New?

• The present study provides the first direct evidence that inhibition of amino acid sensor GCN2 activity by GCN2 gene deletion attenuates pressure overload induced congestive heart failure.

• GCN2 gene deletion increased the expression of the anti-apoptotic factor Bcl-2, while reducing both cardiac oxidative stress and cardiomyocyte apoptosis.

What Is Relevant?

• Congestive heart failure is a major cardiovascular disease of epidemic proportion that has increased in prevalence in the past few decades. The data identify GCN2 as a novel maladaptive factor in heart failure.

Summary

• GCN2 expression is maladaptive in congestive heart failure Strategies to reduce GCN2 activity in cardiac tissue may be a novel approach to attenuate congestive heart failure development.