Hypoxia-Inducible Factor 1-Alpha and Glucose Metabolism during Cardiac Remodeling Progression from Hypertrophy to Heart Failure

In pathological cardiac hypertrophy, the heart is more dependent on glucose than fatty acids. This shift in energy metabolism occurs due to several factors, including the oxygen deficit, which activates hypoxia-inducible factor-1α (HIF-1α), a critical molecule related to glucose metabolism. However, there are gaps regarding the behavior of key proteins in the glycolytic pathway and HIF-1α during the transition from hypertrophy to heart failure (HF). This study assesses the hypothesis that there is an early change and enhancement of HIF-1α and the glycolytic pathway, as well as an association between them during cardiac remodeling. Sham and aortic stenosis Wistar rats were analyzed at 2, 6, and 18 weeks and in HF (n = 10–18). Cardiac structure and function were investigated by echocardiogram. Myocardial glycolysis, the aerobic and anaerobic pathways and glycogen were analyzed by enzymatic assay, Western blot, and enzyme-linked immunosorbent assay (ELISA). The following were observed: increased left ventricular hypertrophy; early diastolic function change and severe systolic and diastolic dysfunction in HF; increased HIF-1α in the 2nd week and in HF; precocious alteration and intensification of glycolysis with a shift to anaerobic metabolism from the 6th week onwards; association between HIF-1α, glycolysis, and the anaerobic pathway. Our hypothesis was confirmed as there was an early change and intensification in glucose metabolism, alteration in HIF-1α, and an association between data during the progression from hypertrophy to heart failure.


Introduction
The heart can meet energy demands using various substrates, such as glucose, lactate, fatty acids, amino acids, and ketone bodies [1,2]. Around 40-70% of the energy needed during the contraction and relaxation processes in the adult heart is usually obtained from fatty acids, with glucose contributing about 20-30% [2,3].
In pathological cardiac hypertrophy, the metabolism is reprogrammed with greater dependence on glucose to the detriment of fatty acids so that the heart reverts to the fetal metabolic condition [1,2]. The mechanisms involved in switching the substrates are not sufficiently clarified, and the classic explanation for this imbalance requires oxygen (O 2 ) deficit [4].
In response to the lack of O 2 and adenosine triphosphate (ATP) [5], protein expression changes occur in different cellular processes [5][6][7][8] to increase tissue O 2 and ATP availability to ensure cell maintenance and survival [5,6]. One of the main factors involved in these events is attributed to hypoxia-inducible factor-1α (HIF-1α), a transcriptional factor responsible for activating hundreds of genes, including those associated with glucose metabolism [9][10][11].
HIF-1α intensifies the glycolytic pathway during the cardiac remodeling (CR) process [12]. Although there is an extensive body of literature on glucose metabolism in different types of heart disease [13][14][15][16][17][18][19], showing an increased reliance on glucose with an overall reduced oxidative metabolism, i.e., a reappearance of the fetal metabolic pattern, there are gaps concerning the involvement of HIF-1α and glucose metabolism during CR progression. This study aimed to evaluate the hypothesis that there is an early change and intensification of HIF-1α, glucose metabolism, and the association between these variables during the progression from hypertrophy to heart failure (HF). Knowing the initial moment of O 2 deficit and metabolic alterations will shed light on the molecular mechanisms underlying CR progression and assist in the development of possible therapeutic strategies in treating heart disease due to chronic pressure overload.

Clinical-Pathological Signs of Heart Failure
All rats in the AoS HF group presented altered breathing patterns, ascites, pleural effusion, right ventricular (RV) hypertrophy, and left atrial thrombus. The left atrium showed a large thrombus in 80% of the rats and a moderate thrombus in 20%; RV hypertrophy was marked in 90% and moderate in 10%. The Sham HF group showed no clinical or pathological signs of HF (Table 1). Table 1. Clinical and pathological data of heart failure (HF).

Body and Lung Weights and Post Mortem Cardiac Morphology
Body weight increased over time and stabilized after the 18th week in both the Sham and Aos groups ( Figure 1A). Left ventricle corrected by tibia (LV/TB) in Aos HF was higher than all other AoS groups; it was more significant in Aos 18 and AoS 6 than AoS 2 . This variable was also higher in Sham HF than Sham 6 and Sham 2 and more prominent in Sham 18 than Sham 2 . LV/TB was higher in AoS groups compared to their respective Shams ( Figure 1B).
RV/TB was higher in Aos HF than the other AoS groups; it was more significant in Aos 18 and AoS 6 than AoS 2 . RV corrected by tibia (RV/TB) was higher in AoS HF and AoS 18 groups compared to their respective Shams ( Figure 1C). Atrium corrected by TB (AT/TB) and lung corrected by TB (Lung/TB) were more prominent in Aos HF than the other AoS groups; these variables were also higher in Aos 18 and AoS 6 than AoS 2 . AT/TB were higher in AoS HF , AoS 18 , and AoS 6 compared to their respective Shams, while Lung/TB was Data are expressed as mean ± SD and analyzed by ANOVA and Tukey. p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments (n = 12-18 animals for each group). RV/TB was higher in AosHF than the other AoS groups; it was more significant in Aos18 and AoS6 than AoS2. RV corrected by tibia (RV/TB) was higher in AoSHF and AoS18 groups compared to their respective Shams ( Figure 1C). Atrium corrected by TB (AT/TB) and lung corrected by TB (Lung/TB) were more prominent in AosHF than the other AoS groups; these variables were also higher in Aos18 and AoS6 than AoS2. AT/TB were higher in AoSHF, AoS18, and AoS6 compared to their respective Shams, while Lung/TB was more prominent in AoSHF than ShamHF ( Figure 1D,E). These data indicate a dysfunctional hypertrophied heart that was accentuated in the HF phase.

Heart Rate, Structural and Functional Echocardiographic Data
Heart rate (HR) was more significant in Sham2 than the other control groups; it was higher in Sham6 than ShamHF. HR in AoS2 animals was lower than in Sham2 (Figure 2A). Left atrium diameter normalized to aortic diameter (LA/AO) ratio was more significant in AoSHF than the other three AoS groups; this ratio was higher in all AoS groups in relation to their respective Sham group ( Figure 2B). Left ventricle diastolic diameter (LVDD) was higher in AoSHF than the other AoS groups; this variable was higher in AoS6 and AoS18 than AoS2. It was also lower in Sham2 than the other Sham groups; LVDD was higher in AoS6, AoS18, and AoSHF in relation to their respective controls ( Figure 2C). Left ventricle posterior wall diastolic thickness (LVWDT) was higher in AoSHF and AoS18 than AoS6 and AoS2; it was also higher in AoS6 than AoS2. In ShamHF and Sham18, it was higher than in Sham6 and Sham2. LVWDT was higher in AoS6, AoS18, and AoSHF in relation to their respective Sham groups ( Figure 2D). Left ventricle relative wall thickness (RWT) was higher in AoS18 and AoSHF than AoS2 and AoS6; it was also higher in Sham2 than Sham6. RWT was higher in AoS6, AoS18, and AoSHF than their respective Sham groups ( Figure 2E).

Heart Rate, Structural and Functional Echocardiographic Data
Heart rate (HR) was more significant in Sham 2 than the other control groups; it was higher in Sham 6 than Sham HF . HR in AoS 2 animals was lower than in Sham 2 ( Figure 2A). Left atrium diameter normalized to aortic diameter (LA/AO) ratio was more significant in AoS HF than the other three AoS groups; this ratio was higher in all AoS groups in relation to their respective Sham group ( Figure 2B). Left ventricle diastolic diameter (LVDD) was higher in AoS HF than the other AoS groups; this variable was higher in AoS 6 and AoS 18 than AoS 2 . It was also lower in Sham 2 than the other Sham groups; LVDD was higher in AoS 6 , AoS 18 , and AoS HF in relation to their respective controls ( Figure 2C). Left ventricle posterior wall diastolic thickness (LVWDT) was higher in AoS HF and AoS 18 than AoS 6 and AoS 2 ; it was also higher in AoS 6 than AoS 2 . In Sham HF and Sham 18 , it was higher than in Sham 6 and Sham 2 . LVWDT was higher in AoS 6 , AoS 18 , and AoS HF in relation to their respective Sham groups ( Figure 2D). Left ventricle relative wall thickness (RWT) was higher in AoS 18 and AoS HF than AoS 2 and AoS 6 ; it was also higher in Sham 2 than Sham 6 . RWT was higher in AoS 6 , AoS 18 , and AoS HF than their respective Sham groups ( Figure 2E). Echocardiographic data show hypertrophy development over time in animals who underwent AoS surgery.
Analysis of systolic function showed that ejection fraction (EF) decreased in AoS HF compared to the other AoS groups; it was higher in AoS 6 than Sham 6 and lower in AoS HF than Sham HF ( Figure 3A). Mesocardial shortening (Meso) was lower in AoS HF than the other AoS groups; in AoS 18 it was lower than AoS 6 and AoS 2 . It was higher in AoS 6 than Sham 6 and lower in AoS HF than Sham HF ( Figure 3B). Posterior wall shortening velocity (PWSV) values were lower in AoS 18 and AoS HF than their respective Shams ( Figure 3C).
Echocardiographic data show hypertrophy development over time in animals who underwent AoS surgery. Analysis of systolic function showed that ejection fraction (EF) decreased in AoSHF compared to the other AoS groups; it was higher in AoS6 than Sham6 and lower in AoSHF than ShamHF ( Figure 3A). Mesocardial shortening (Meso) was lower in AoSHF than the other AoS groups; in AoS18 it was lower than AoS6 and AoS2. It was higher in AoS6 than Sham6 and lower in AoSHF than ShamHF ( Figure 3B). Posterior wall shortening velocity (PWSV) values were lower in AoS18 and AoSHF than their respective Shams ( Figure 3C).  Analysis of diastolic function showed that E wave was higher in AoSHF than the other AoS groups; it was more significant in AoS6 and AoS18 than AoS2. Early diastolic mitral inflow velocity (E wave) was higher in AoS6, AoS18, and AoSHF compared to their respective Sham groups ( Figure 3D). The late diastolic mitral inflow velocity (A wave) was smaller in AoSHF than the other AoS groups; it was higher in AoS6 and AoS18 compared to the respective Shams and lower in AoSHF than ShamHF ( Figure 3E). The E/A ratio was Analysis of diastolic function showed that E wave was higher in AoS HF than the other AoS groups; it was more significant in AoS 6 and AoS 18 than AoS 2 . Early diastolic mitral inflow velocity (E wave) was higher in AoS 6 , AoS 18 , and AoS HF compared to their respective Sham groups ( Figure 3D). The late diastolic mitral inflow velocity (A wave) was smaller in AoS HF than the other AoS groups; it was higher in AoS 6 and AoS 18 compared to the respective Shams and lower in AoS HF than Sham HF ( Figure 3E). The E/A ratio was higher in AoS HF compared to the other AoS groups; it was also higher in AoS HF than Sham HF ( Figure 3F). Function analysis by echocardiogram showed that animals with aortic stenosis had early diastolic dysfunction which was accentuated with the evolution of RC and was accompanied by a decrease in systolic function in the HF phase.

Cardiac HIF-1α Expression
Analysis of evolution in animals with heart disease showed that HIF-1α was higher in AoS HF than AoS 2 and AoS 18 . The difference of 2.58% between AoS 6 vs. AoS HF (24.17%) and AoS 18 vs. AoS HF (26.76%) groups shows that, although there was a statistical difference in AoS 6 and AoS 18 vs. AoS HF , from a biological point of view their behavior seemed similar. There was a statistical difference in all AoS animals compared to their respective Shams ( Figure 4). The increase in HIF-1α in AoS groups in relation to their respective controls may suggest myocardial hypoxia. Studies on the quantification of cardiac oxygen rate are necessary for further clarification.

Blood Glucose and Serum Insulin
Glucose levels were lower in AoSHF than AoS6 and AoS18, suggesting that time may interfere with the glycemic level ( Figure 5A). Insulin levels increased in AoS6 and AoS18 compared to AoS2; however, they decreased in AoSHF compared to AoS6 and AoS18. Insulin was higher in AoS6 and AoS18 than their respective Shams; it was significantly lower in AoSHF compared to ShamHF ( Figure 5B). Insulin concentrations changed during the course of CR; the reduction in the AoSHF phase may be due to the inefficiency in pancreatic cells to produce insulin.

Blood Glucose and Serum Insulin
Glucose levels were lower in AoS HF than AoS 6 and AoS 18 , suggesting that time may interfere with the glycemic level ( Figure 5A). Insulin levels increased in AoS 6 and AoS 18 compared to AoS 2 ; however, they decreased in AoS HF compared to AoS 6 and AoS 18 . Insulin was higher in AoS 6 and AoS 18 than their respective Shams; it was significantly lower in AoS HF compared to Sham HF ( Figure 5B). Insulin concentrations changed during the course of CR; the reduction in the AoS HF phase may be due to the inefficiency in pancreatic cells to produce insulin. difference between moments (n = 8 animals for each group).

Blood Glucose and Serum Insulin
Glucose levels were lower in AoSHF than AoS6 and AoS18, suggesting that time may interfere with the glycemic level ( Figure 5A). Insulin levels increased in AoS6 and AoS18 compared to AoS2; however, they decreased in AoSHF compared to AoS6 and AoS18. Insulin was higher in AoS6 and AoS18 than their respective Shams; it was significantly lower in AoSHF compared to ShamHF ( Figure 5B). Insulin concentrations changed during the course of CR; the reduction in the AoSHF phase may be due to the inefficiency in pancreatic cells to produce insulin.

Cardiac Insulin Signaling and Glucose Transporters 1 and 4
With regard to phosphorylated insulin receptor (pIR) level, it was higher in AoS HF than the other AoS groups, higher in AoS 18 and AoS 6 than AoS 2 , and more significant in AoS HF , AoS 18 , and AoS 6 than their respective Shams ( Figure 6A). Total insulin receptor (IR) increased in AoS 6 and AoS 18 compared to their respective Shams ( Figure 6B). The pIR/Total IR ratio was higher in AoS HF than the other AoS groups; in AoS 18 and AoS 6 it was higher than AoS 2 ; it was higher in Sham HF and Sham 6 than Sham 18 and Sham 2 . It was more significant in AoS HF , AoS 18 , and AoS 6 than their respective Shams ( Figure 6C). With regard to phosphorylated protein kinase B (pAKT), it was higher in AoS HF than AoS 6 and AoS 2 ; higher in AoS 18 and Sham HF than AoS 2 ; and it was also higher in AoS HF than Sham HF ( Figure 6D). Total protein kinase B (AKT) values in AoS HF and AoS 18 were higher than AoS 6 and AoS 2 ; they were higher in Sham HF and Sham 18 than Sham 2 ; and higher in AoS HF than Sham HF ( Figure 6E). The pAKT/Total AKT ratio was higher in AoS HF than AoS 2 ; this was more significant in AoS HF than Sham HF ( Figure 6F). Phosphoinositide 3-kinase (PI3K) was higher in AoS HF than the other AoS groups; it was more significant in AoS HF than Sham HF ( Figure 6G). Glucose transporters 4 (GLUT4) was higher in AoS 6 , AoS 18 , and AoS HF compared to their respective Shams ( Figure 6H). Glucose transporters 1 (GLUT1) was higher in AoS HF than the other AoS groups; it was more significant in AoS groups then their respective Shams ( Figure 6I). These results show activation of the insulin pathway and increased GLUT1 expression, accentuated in the heart failure phase. These changes, which could result in increased glucose uptake, are aimed at maintaining hypertrophied heart energy demand. Studies to quantify cardiac glucose uptake are necessary for further clarification.

Cardiac Glycolysis and Glycogen
Hexokinase II (HKII) was higher in AoSHF than the other AoS animals; it was higher in AoS18 and AoS6 than AoS2. It was also more significant in AoSHF, AoS18, and AoS6 than their respective Shams ( Figure 7A). HKII activity was higher in AoSHF than the other AoS groups; it was also lower in Sham18 and Sham6 than Sham2. It was also more significant in all AoS groups than their respective Shams ( Figure 7B). 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFK2) was higher in AoSHF than the other AoS groups; it was

Cardiac Glycolysis and Glycogen
Hexokinase II (HKII) was higher in AoS HF than the other AoS animals; it was higher in AoS 18 and AoS 6 than AoS 2 . It was also more significant in AoS HF , AoS 18 , and AoS 6 than their respective Shams ( Figure 7A). HKII activity was higher in AoS HF than the other AoS groups; it was also lower in Sham 18 and Sham 6 than Sham 2 . It was also more significant in all AoS groups than their respective Shams ( Figure 7B). 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFK2) was higher in AoS HF than the other AoS groups; it was higher in AoS 6 , AoS 18, and AoS HF than their respective Shams ( Figure 7C). Pyruvate kinase (PK) was higher in AoS HF than the other AoS groups; it was lower in Sham 2 than the other control groups. PK was higher in all AoS groups than their respective Shams ( Figure 7D). Over time, cardiac glycogen exhibited lower concentration in AoS HF than AoS 2 , AoS 6 , and AoS 18 ; concentration was elevated in AoS 6 compared to AoS 2 and AoS 18 . Cardiac glycogen was higher in AoS 6 and AoS 18 and lower in AoS HF than their respective Shams ( Figure 7E). Generally, the glycolysis pathway increases in HF to increase energy for the heart. In the most severe phase of the disease, glycogen stores are reduced, either by insufficient storage or by greater use of this substrate than exogenous glucose. higher in AoS6, AoS18, and AoSHF than their respective Shams ( Figure 7C). Pyruvate kinase (PK) was higher in AoSHF than the other AoS groups; it was lower in Sham2 than the other control groups. PK was higher in all AoS groups than their respective Shams ( Figure 7D). Over time, cardiac glycogen exhibited lower concentration in AoSHF than AoS2, AoS6, and AoS18; concentration was elevated in AoS6 compared to AoS2 and AoS18. Cardiac glycogen was higher in AoS6 and AoS18 and lower in AoSHF than their respective Shams ( Figure 7E). Generally, the glycolysis pathway increases in HF to increase energy for the heart. In the most severe phase of the disease, glycogen stores are reduced, either by insufficient storage or by greater use of this substrate than exogenous glucose.

Cardiac Aerobic and Anaerobic Pathway
Pyruvate dehydrogenase (PDH) expression was lower in AoSHF than the other AoS animals; it was lower in AoSHF and AoS18 than their respective Shams ( Figure 8A). Citrate synthase (CS) activity was lower in AoSHF than the other AoS animals; it was lower in AoS6, AoS18, and AoSHF compared to their respective Shams ( Figure 8B). Lactate dehydrogenase (LDH) expression increased in AoSHF and AoS18 than in their respective Shams ( Figure 8C). LDH activity was more significant in AoS6, AoS18, and AoSHF than AoS2; it was more significant in AoS6, AoS18, and AoSHF than their respective Shams ( Figure 8D). Lactate levels were more significant in AoS6, AoS18, and AoSHF than AoS2; they was higher in Sham6 than ShamHF. They were also more significant in AoS6, AoS18, and AoSHF than their respective Shams ( Figure 8E). Our results show deviation from the aerobic to anaerobic pathway in hypertrophied myocardium of rats with AoS. The data associated with the increase in HIF-1α suggests an O2 deficit in cardiac tissue that arises during the remodeling process.

Cardiac Aerobic and Anaerobic Pathway
Pyruvate dehydrogenase (PDH) expression was lower in AoS HF than the other AoS animals; it was lower in AoS HF and AoS 18 than their respective Shams ( Figure 8A). Citrate synthase (CS) activity was lower in AoS HF than the other AoS animals; it was lower in AoS 6 , AoS 18 , and AoS HF compared to their respective Shams ( Figure 8B). Lactate dehydrogenase (LDH) expression increased in AoS HF and AoS 18 than in their respective Shams ( Figure 8C). LDH activity was more significant in AoS 6 , AoS 18 , and AoS HF than AoS 2 ; it was more significant in AoS 6 , AoS 18 , and AoS HF than their respective Shams ( Figure 8D). Lactate levels were more significant in AoS 6 , AoS 18 , and AoS HF than AoS 2 ; they was higher in Sham 6 than Sham HF . They were also more significant in AoS 6 , AoS 18 , and AoS HF than their respective Shams ( Figure 8E). Our results show deviation from the aerobic to anaerobic pathway in hypertrophied myocardium of rats with AoS. The data associated with the increase in HIF-1α suggests an O 2 deficit in cardiac tissue that arises during the remodeling process. Figure 9 shows the overview results obtained from HIF-1α and glucose metabolism during the evolution process of cardiac remodeling due to pressure overload.  Figure 9 shows the overview results obtained from HIF-1α and glucose metabolism during the evolution process of cardiac remodeling due to pressure overload.   Figure 9 shows the overview results obtained from HIF-1α and glucose metabolism during the evolution process of cardiac remodeling due to pressure overload.

Canonical Correlation Analysis (CCA)
The primary objective of CCA is to measure a linear association between sets of variables. In this study, we measured the association between HIF-1α and the following ensemble derived from (a) insulin signaling and glucose transporters 1 and 4 (pIR, Total IR, pAKT, Total AKT, PI3K, GLUT1, and GLUT4 expressions; (b) glycolysis (HKII activity and HKII, PFK2 and PK expressions); (c) anaerobic pathway (LDH activity and LDH expression); and (d) aerobic pathway (CS activity and PDH expression). There was a positive linear association between canonical variable u1 (HIF-1α) and ν1 ( Figure 10A-C). In this sense, u1 presents a high association with ν1, mainly in the AoS HF group, and a low association in the AoS 2 group. AoS 6 and AoS 18 animals have dispersed behavior around the average. There was no significant association between HIF-1α and the aerobic pathway parameters ( Figure 10D). Different symbols mean statistical difference between AoS vs. Sham (↑ increase or ↓ decrease) or between the AoS groups ( † vs. AoS2; # vs. Aos6; *** vs. Aos2, 6, 18; ** vs. Aos 2, 18; * vs. Aos6, 18; Ω vs. Aos2, 6), p < 0.05.

Canonical Correlation Analysis (CCA)
The primary objective of CCA is to measure a linear association between sets of variables. In this study, we measured the association between HIF-1α and the following ensemble derived from (a) insulin signaling and glucose transporters 1 and 4 (pIR, Total IR, pAKT, Total AKT, PI3K, GLUT1, and GLUT4 expressions; (b) glycolysis (HKII activity and HKII, PFK2 and PK expressions); (c) anaerobic pathway (LDH activity and LDH expression); and (d) aerobic pathway (CS activity and PDH expression). There was a positive linear association between canonical variable 1 (HIF-1α) and 1 (Figure 10A-C). In this sense, 1 presents a high association with 1, mainly in the AoSHF group, and a low association in the AoS2 group. AoS6 and AoS18 animals have dispersed behavior around the average. There was no significant association between HIF-1α and the aerobic pathway parameters ( Figure 10D).

Discussion
This study analyzed HIF-1α, the glucose metabolism, aerobic and anaerobic pathways, and the association between HIF-1α and variables during the transition from compensated hypertrophy to HF. To our knowledge, this is the first study to show heart disease progression regarding this major hypoxia marker and glucose metabolism.
We found that HIF-1α increased in all groups with heart disease, with an important increase during CR progression, especially in the HF phase. High levels of HIF-1α, resulting from a decrease in its degradation in the presence of tissue hypoxia [5,[20][21][22], may be due to the imbalance between the supply and consumption of O 2 by the myocardium due to disharmony in the proliferative capacity of the capillary network, vascular rarefaction, the degree of concentric hypertrophy, and myocardial fibrosis [23][24][25][26][27]. The accentuation seen over time of these myocardial structural changes may have been responsible for increasing HIF-1α in the different phases of aortic stenosis.
Although HIF-1α has a cardioprotective action by restoring energy homeostasis [22,28], there is evidence that persistent elevation, as observed in the AoS 6 , AoS 18 and AoS HF groups, may participate in the genesis of hypertrophy [12] along with other factors such as parietal mechanical stress and the neuroendocrine system [27,[29][30][31][32]. In addition to action in the hypertrophic process, HIF-1α can also participate in the genesis of ventricular dysfunction observed in cardiac groups, as it causes a suppression of/decrease in sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a) synthesis [33,34] or fibrosis development [35,36]. Other factors may also have participated in this dysfunction, such as apoptosis, reactivation of fetal gene expression, altered sarcomere structure, insufficient angiogenesis, mitochondrial dysfunction, impairment of the cellular Ca 2+ transient, and metabolic reprogramming [26,27,31,37]. Although there are investigations that have shown increased HIF-1α in hypertrophy related to heart failure [11], to our knowledge no other studies have systematically evaluated HIF-1α behavior during the different phases of cardiac remodeling evolution due to overload mechanics.
GLUT1 increased in all groups with pressure overload which was accentuated during cardiac remodeling, mainly in the HF phase. GLUT1 is a glucose transporter, independent of insulin, responsible for glucose uptake in the fetal stage [38]. Although its expression reduces after birth, GLUT1 can be reinduced in cardiac patients [39]. The early rise in GLUT1 indicates an attempt to increase glucose uptake by the myocardium from the initial aggression phase (AoS 2 ). As HIF-1α is a transcription factor for GLUT1 [28,40], and it increased during the evolutionary process, we can suggest that HIF-1α may be involved in the GLUT1 elevation observed in this study. Other potential factors implied in increasing this transporter are hypertrophic stimuli, such as alpha-adrenergic agonist and ischemia/hypoxia [38,41]. Our results are consistent with the literature, which shows elevated GLUT1 during pathological hypertrophy [27,38,42].
Insulin signaling pathway activity rose in all aortic stenosis groups from the sixth week onwards, increasing over time with the exception of AoS HF which showed a drop in insulin level. Hyperinsulinemia in AoS 6 and AoS 18 , consistent with insulin resistance in cardiac patients [15,43], aims to maintain glucose homeostasis at the expense of elevating this hormone. Increased insulin may have promoted a rise in IR phosphorylation but was not accompanied by an elevation in main insulin signaling pathway proteins, PI3K and AKT. Once that this cascade had been compromised in the cardiac groups, other factors might have increased sarcolemmal GLUT4, such as ischemia and cardiac contraction [38]. In AoS HF , in contrast to AoS 6 and AoS 18 groups, the hypoinsulinemia may have occurred from the pancreas being unable to produce insulin probably due to structural and functional changes and decreased food intake in animals with HF [44]. In hypoinsulinemia, increased expression of pIR, Total IR, PI3K, pAKT, and Total AKT may be aimed at maintaining adequate glucose uptake for the hypertrophied myocardium to attenuate energy dysregulation in an attempt to prolong cell survival. The mechanisms responsible for the increase in glucose uptake proteins in hypoinsulinemia in heart failure need to be clarified. According to the literature, in pathological hypertrophy, GLUT4 levels are reduced [27,38,42] or in-creased [45]; our results are in agreement with Cook and collaborators [45] as we found a rise in and stabilization of GLUT4 after the sixth week of aortic stenosis. With regard to the insulin/IR/PI3K/AKT signaling pathway, the literature shows an increase in these variables in pressure-overload-induced cardiac animals [37], corroborating our findings, except for insulin in the HF phase.
The glycolytic pathway showed an increase in all cardiopathy groups; this intensified during remodeling and was more expressive in the HF period. AoS 2 animals, in which left ventricle (LV) hypertrophy was less pronounced than the other AoS groups, showed increased HKII activity and PK expression. HKII enzyme phosphorylates glucose into glucose 6 phosphate (G-6P) to prevent glucose release from inside the cell. Its activity is inhibited by high G-6P and ATP concentrations and stimulated by high levels of adenosine diphosphate, adenosine monophosphate, and phosphate [46]; therefore, the presumed increase in these factors by hypoxia in our study could have caused a rise in HKII enzyme activity in all cardiopathy groups.
PK is responsible for producing pyruvate and ATP from the phosphoenolpyruvate substrate [47]. Different factors are involved in the increased expression of this enzyme, e.g., HIF-1α [48]; since this transcriptional factor is high in AoS 2 , it could be responsible for raising PK expression. In AoS 6 , AoS 18 , and AoS HF , in addition to the changes shown in AoS 2 , there were novel alterations, such as increased HKII and PFK2 expression, possibly due to greater energy demand; the factors involved in raising these glycolytic enzymes may be HIF-1α and insulin [49,50]. These two elements might have participated until the 18th week; since there was a decay in insulin in the HF phase, HIF-1α may have been largely responsible for these enzyme changes. Intensification of the glycolytic pathway throughout CR progression, mainly in the HF phase, displays an attempt to compensate for the increasing energy deficit resulting from the decline in O 2 supply. According to the literature, glycolysis is elevated in different animal models of cardiac hypertrophy [51], corroborating our results. However, to our knowledge, no study has evaluated the glycolytic pathway during CR progression due to pressure overload.
Although glycolysis was exacerbated, an increase in cardiac glycogen storage was seen in AoS 6 and AoS 18 and a robust reduction seen in AoS HF animals. This elevation signals that glycogen synthesis was more significant than glycogenolysis, probably due to the increase in exogenous glucose uptake by the increased translocation of GLUT 1 and 4 ( Figure 6H,I) and glycogen synthase kinase 3β (GSK-3β) inhibition [31]. During CR progression, the glycogen decay after the 18th week, which intensified in the HF phase, indicates that hypertrophied hearts started to preferentially use glucose from glycogen stores as opposed to exogenous glucose; this is possibly due to increased energy demand and reduced insulin or enzyme activity involved in glycogenesis [52]. According to the literature, glycogen synthesis is elevated in hypertrophied hearts, corroborating our findings [53]. We did not find any study that assessed myocardial glycogen levels in severe heart failure.
The anaerobic and aerobic pathways changed in cardiopathy groups from the sixth week onwards with an increase in LDH activity and lactate concentration, and reduced CS activity; also, LDH expression rose in AoS 18 and PDH expression decreased in AoS HF . These modifications indicate a shift from the aerobic to anaerobic pathway, possibly by tissue hypoxia, i.e., lack of myocardial O 2 due to mitochondrial dysfunction and disharmony between the capillary network and degree of hypertrophy [17,54,55]. This deviation towards the anaerobic pathway suggests that although there was an increase in glycolysis, it may not have been accompanied by a rise in glucose oxidation [38]. Interestingly, although the aerobic and anaerobic pathways displayed concomitant changes in the sixth week, during evolution of the hypertrophic process, the anaerobic route remained stable. In contrast, the aerobic pathway showed a marked decline in the HF phase. Our findings agree with data from the literature observed in different cardiac hypertrophy models [53,56]. To our knowledge, there is no information regarding the moment when the aerobic pathway changes to anaerobic during CR progression due to pressure overload.
The canonical association data revealed that HIF-1α had a positive and significant linear correlation with the sets of variables that analyzed insulin signaling and GLUT 1 and GLUT 4, glycolysis, and the anaerobic pathway. The most intense correlations from each group occurred with proteins that are expressed by transcriptional factor HIF-1α, such as GLUT1, PFK2, and LDH [11,28,57]. Although these associations do not indicate a cause-andeffect relationship, their significance shows that HIF-1α may be involved in gene alterations during the process of glycidic metabolic remodeling in this experimental model. These results have biological support in the literature, where HIF-1α is described as participating in the transcription of proteins involved in glucose metabolism. We suggest further studies that perform HIF-1α blocking to understand the cause-and-effect relationship of this protein and its action on the metabolism and function of the remodeled heart. This and other concerns observed in this work can only be obtained through additional investigation into HIF-1α, energy metabolism, and heart remodeling.

Animals, Supravalvar Surgery, and Experimental Groups
Twenty-one-day-old male Wistar rats were housed in individual cages with controlled temperature (24 ± 2 • C), humidity (55 ± 5%), and light (12 h light/dark cycle), and free access to food and water. All experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the National Research Council (2011) and approved by the Ethics Committee on Animals Experiments of the Botucatu Medical School, São Paulo State University, UNESP (1081/2014-CEUA).

Body and Lung Weights and Post Mortem Cardiac Morphology
The final body weight (BW), lung weight, and lung/tibia ratio were measured at the end of each experimental period. Cardiac hypertrophy was determined by macroscopic analysis of the following parameters: atrium (AT), LV, and RV weights, as well as their ratio with tibia length.

Cardiac Glycogen
Cardiac glycogen content was determined as described by Trinder (1969) [67]. The test medium consisted of 30 mM phosphate buffer, 1 mM phenol, 12.5U glucose oxidase, 0.8U peroxidase, 0.29 mM 4-aminoantipyrine, and 0.05% Triton X-100. Total assay volume was 165 µL, and 20 µL of homogenate was added. Absorbance delta was monitored at 505 nm. Corrected values were subtracted from the control well reading (without amyloglucosidase) and from the reaction well reading (with amyloglucosidase) for each sample. Glucose content was determined using a glucose standard.

Statistical Analysis
Data are expressed as mean ± SD (standard deviation) or median (minimum [Min] and maximum [Max] values). Echocardiographic, cardiac metabolic, and structural variables, considering both sources of variation, i.e., AoS (absence or presence) and euthanasia moment (2, 6, 18 weeks and HF) were analyzed by ANOVA using a 2 × 4 factorial scheme with a completely randomized design; this was complemented with Tukey's multiple comparisons test (for variables with adherence normality) and non-parametric analysis of variance technique (ANOVANP) followed by Dunn's post hoc test (for variables with a lack of adherence normality). The choice of parametric or non-parametric procedure was based on the Kolmogorov-Smirnov normality test [68]. Regarding the study of the linear association between HIF-1 and the set of metabolic variables, the exploratory multivariate data technique involving the canonical correlation was used [69]. All statistical conclusions were discussed at a 5% significance level. The level of significance considered was 5%. Analyses were performed using RStudio version 1.1.442 (Boston, MA, USA), and graphics were generated using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
In conclusion, our hypothesis was confirmed as there was an early change and intensification of the glucose metabolism, observed by increased activity and/or expression of glucose transporters, glycolysis enzymes, and proteins involved in lactate production and the intracellular insulin signaling cascade, concomitant with high levels of HIF-1α during the progression from hypertrophy to heart failure.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data were included in this manuscript. Additional datasets generated during and/or analyzed during the current study are available from the corresponding author upon request.