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

Abstract

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.

1. 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 (O₂) deficit [4].

In response to the lack of O₂ and adenosine triphosphate (ATP) [5], protein expression changes occur in different cellular processes [5,6,7,8] to increase tissue O₂ 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 O₂ 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.

2. Results

2.1. 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).

2.2. 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 Aos₁₈ and AoS₆ than AoS₂. This variable was also higher in Sham_HF than Sham₆ and Sham₂ and more prominent in Sham₁₈ than Sham₂. 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 Aos₁₈ and AoS₆ than AoS₂. RV corrected by tibia (RV/TB) was higher in AoS_HF 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 Aos_HF than the other AoS groups; these variables were also higher in Aos₁₈ and AoS₆ than AoS₂. AT/TB were higher in AoS_HF, AoS₁₈, and AoS₆ compared to their respective Shams, while Lung/TB was more prominent in AoS_HF than Sham_HF (Figure 1D,E). These data indicate a dysfunctional hypertrophied heart that was accentuated in the HF phase.

2.3. 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 Sham_HF. 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 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 AoS₆ and AoS₁₈ than AoS₂. It was also lower in Sham₂ than the other Sham groups; LVDD was higher in AoS₆, AoS₁₈, and AoS_HF in relation to their respective controls (Figure 2C). Left ventricle posterior wall diastolic thickness (LVWDT) was higher in AoS_HF and AoS₁₈ than AoS₆ and AoS₂; it was also higher in AoS₆ than AoS₂. In Sham_HF and Sham₁₈, it was higher than in Sham₆ and Sham₂. LVWDT was higher in AoS₆, AoS₁₈, and AoS_HF in relation to their respective Sham groups (Figure 2D). Left ventricle relative wall thickness (RWT) was higher in AoS₁₈ and AoS_HF than AoS₂ and AoS₆; it was also higher in Sham₂ than Sham₆. RWT was higher in AoS₆, AoS₁₈, 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 AoS₆ than Sham₆ and lower in AoS_HF than Sham_HF (Figure 3A). Mesocardial shortening (Meso) was lower in AoS_HF than the other AoS groups; in AoS₁₈ it was lower than AoS₆ and AoS₂. It was higher in AoS₆ than Sham₆ and lower in AoS_HF than Sham_HF (Figure 3B). Posterior wall shortening velocity (PWSV) values were lower in AoS₁₈ and AoS_HF than their respective Shams (Figure 3C).

Analysis of diastolic function showed that E wave was higher in AoS_HF 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 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 AoS₆ and AoS₁₈ 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.

2.4. Cardiac HIF-1α Expression

Analysis of evolution in animals with heart disease showed that HIF-1α was higher in AoS_HF than AoS₂ and AoS₁₈. The difference of 2.58% between AoS₆ vs. AoS_HF (24.17%) and AoS₁₈ vs. AoS_HF (26.76%) groups shows that, although there was a statistical difference in AoS₆ and AoS₁₈ 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.

2.5. Blood Glucose and Serum Insulin

Glucose levels were lower in AoS_HF 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 AoS_HF compared to AoS₆ and AoS₁₈. Insulin was higher in AoS₆ and AoS₁₈ 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.

2.6. 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 AoS₁₈ and AoS₆ than AoS₂, and more significant in AoS_HF, AoS₁₈, and AoS₆ than their respective Shams (Figure 6A). Total insulin receptor (IR) increased in AoS₆ and AoS₁₈ compared to their respective Shams (Figure 6B). The pIR/Total IR ratio was higher in AoS_HF than the other AoS groups; in AoS₁₈ and AoS₆ it was higher than AoS₂; it was higher in Sham_HF and Sham₆ than Sham₁₈ and Sham₂. It was more significant in AoS_HF, AoS₁₈, and AoS₆ than their respective Shams (Figure 6C). With regard to phosphorylated protein kinase B (pAKT), it was higher in AoS_HF than AoS₆ and AoS₂; higher in AoS₁₈ and Sham_HF than AoS₂; and it was also higher in AoS_HF than Sham_HF (Figure 6D). Total protein kinase B (AKT) values in AoS_HF and AoS₁₈ were higher than AoS₆ and AoS₂; they were higher in Sham_HF and Sham₁₈ than Sham₂; and higher in AoS_HF than Sham_HF (Figure 6E). The pAKT/Total AKT ratio was higher in AoS_HF than AoS₂; 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 AoS₆, AoS₁₈, 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.

2.7. Cardiac Glycolysis and Glycogen

Hexokinase II (HKII) was higher in AoS_HF than the other AoS animals; it was higher in AoS₁₈ and AoS₆ than AoS₂. It was also more significant in AoS_HF, AoS₁₈, and AoS₆ than their respective Shams (Figure 7A). HKII activity was higher in AoS_HF 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 AoS_HF than the other AoS groups; it was higher in AoS₆, 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 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 AoS_HF 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 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.

2.8. 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 AoS₁₈ than their respective Shams (Figure 8A). Citrate synthase (CS) activity was lower in AoS_HF than the other AoS animals; it was lower in AoS₆, AoS₁₈, and AoS_HF compared to their respective Shams (Figure 8B). Lactate dehydrogenase (LDH) expression increased in AoS_HF and AoS₁₈ than in their respective Shams (Figure 8C). LDH activity was more significant in AoS₆, AoS₁₈, and AoS_HF than AoS₂; it was more significant in AoS₆, AoS₁₈, and AoS_HF than their respective Shams (Figure 8D). Lactate levels were more significant in AoS₆, AoS₁₈, and AoS_HF than AoS₂; they was higher in Sham₆ than Sham_HF. They were also more significant in AoS₆, AoS₁₈, 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 O₂ 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.

2.9. 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 𝑣1 (Figure 10A–C). In this sense, 𝑢1 presents a high association with 𝑣1, mainly in the AoS_HF 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).

3. 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 O₂ 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 AoS₆, AoS₁₈ 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 Ca²⁺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 Ca²⁺ 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 (AoS₂). 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 AoS₆ and AoS₁₈, 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 AoS₆ and AoS₁₈ 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 increased [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. AoS₂ 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 AoS₂, it could be responsible for raising PK expression. In AoS₆, AoS₁₈, and AoS_HF, in addition to the changes shown in AoS₂, 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 O₂ 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 AoS₆ and AoS₁₈ 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 AoS₁₈ 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 O₂ 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-and-effect 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.

4. Materials and Methods

4.1. 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).

Supravalvular aortic stenosis (AoS) in young rats has been used as an experimental model to study chronic HF, initially with preserved ejection fraction which may progress to cardiac systolic insufficiency [23,58,59,60]. Ventricular dysfunction occurs gradually, mimicking the development of heart disease in humans. AoS was induced surgically as previously described [23,61,62]. Briefly, a silver clip (0.60 mm internal diameter) was placed on the ascending aorta approximately 3 mm from its root (the AoS group; n = 56). Sham rats underwent the same surgery but without aortic banding (n = 54). Animals from both groups were evaluated at 2, 6, and 18 weeks after surgery and when they presented signs of severe HF, 28–30th week [63,64]. Mortality percentages were as follows: AoS₂ (0%), AoS₆ (17%), AoS₁₈ (27%), AoS_HF (36%). There were no deaths in the Sham group. The following subgroups were formed: Sham₂ (n = 12), Sham₆ (n = 12), Sham₁₈ (n = 15), Sham_HF (n = 15), AoS₂ (n = 12), AoS₆ (n = 14), AoS₁₈ (n = 16), and AoS_HF (n = 14). Rats were observed daily to identify clinical signs of HF: tachypnea, apathetic behavior, altered hair, and loss of muscle mass. The diagnosis was pathologically confirmed post mortem by analyzing the presence and magnitude [(−) absent; (+) light; (++) moderate; (+++) severe] of the following signs: RV hypertrophy, left atrial thrombus, pleural effusion, hemorrhagic liver, and ascites [23]. Animals in the AoS_HF group presented all the signs that characterized severe HF. At the end of each experimental period (2, 6, 18 weeks and HF), animals were fasted for 8 h and were anesthetized with a mixture of ketamine (50 mg/kg, intraperitoneal [i.p.]) and xylazine (10 mg/kg, i.p.) and euthanized by decapitation.

4.2. 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.

4.3. Structural and Functional Echocardiographic Data

Echocardiogram was performed before each euthanasia using a commercially available echocardiograph (General Electric Medical Systems, Vivid S6, Tirat Carmel, Israel) equipped with a 5–11.5 MHz multifrequency transducer as previously described [23,58,59,61]. The following structural variables were analyzed: LA/AO, LVDD, LVWDT, and RWT. The following parameters assessed LV function: percentage of Meso, percentage of EF, PWSV, E and A waves, and E/A ratio.

4.4. Serum Glucose and Insulin Levels

Blood samples were collected from the tail tip before euthanasia for glucose analysis and were assessed using a handheld glucometer (Accu-Chek Go Kit; Roche Diagnostic Brazil Ltd., São Paulo, Brazil). Fasting serum insulin concentrations were measured in blood samples collected after euthanasia and centrifuged at 1620× g for 10 min at 4 °C using an enzyme-linked immunosorbent assay (ELISA) kit (EMD Millipore Corporation, Billerica, MA, USA), according to manufacturer instructions.

4.5. Cardiac HIF-1α and Glucose Metabolism

Cardiac expression of HIF-1α and the proteins involved in glucose metabolism were evaluated by Western blot. LV tissue samples were homogenized in cold RIPA lysis buffer (Amresco, Solon, OH, USA) containing protease (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase (Roche Diagnostics, Indianapolis, IN, USA) inhibitors. The homogenate was centrifuged at 12,000× g for 20 min at 4 °C and supernatant collected. For the extraction of GLUT1 and GLUT4, protein membranes were homogenized in extraction buffer containing 1M Tris-HCl, 25 mM pentetic acid (DTPA), β-Mercaptoethanol, 0.1 M Phenylmethylsulfonyl Fluoride (PMSF), and protease inhibitor. The homogenate was centrifuged at 1000× g for 3 min at 4 °C to remove insoluble material. The supernatant was then centrifuged at 18,000× g for 10 min at 4 °C and transferred to an ultracentrifuge (Sorvall WX80 Ultracentrifuge—Thermo Scientific, Waltham, MA, USA) at 100,000× g for 45 min at 4 °C. After ultracentrifugation, the pellet was resuspended in 100 μL of cold RIPA lysis buffer. Protein concentrations were determined using a Pierce BCA Protein Assay kit (Thermo Scientific, Wilmington, DE, USA). Samples (50 μg) were subjected to SDS-PAGE in polyacrylamide gels (6% or 10% depending on protein molecular weight). After electrophoresis, proteins were electrotransferred to nitrocellulose membrane (Armsham Biosciences, Piscataway, NJ, USA). The blotted membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/Tween-20 (25 mM Tris, pH 7.5, 140 mM sodium chloride, 3mM potassium chloride, and 0.1% Tween-20) for 2 h at room temperature. Membranes were then incubated overnight at 4 °C–8 °C with primary antibody against: HIF-1α (1:500; Abcam, Cambridge, MA, USA; #ab463); IR (1:1000; Cell Signaling, Danvers, MA, USA; #3025); pIR (Tyr1150/1151; 1:1000; Cell Signaling; #3024); PI3K (1:1000; Cell Signaling; #4292); AKT (1:500; Santa Cruz, Delaware Ave, Albany, NY, USA; # sc-5298); pAKT (Thr 308; 1:500; Santa Cruz; #sc-16646-R); HKII (1:1000; Cell Signaling; #2106); PFK2 (1:1000; Millipore, Temecula, CA, USA; #07-1530); PK (1:1000; Cell Signaling; #3186); LDH (1:1000; Cell Signaling; #2012); PDH (1:1000; Cell Signaling; #2784); GLUT1 (1:1000; Cell Signaling; #12939); and GLUT4 (1:1000; Cell Signaling; #2213). Primary antibody binding was detected using peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit IgG, depending on the protein; 1:5000–1:10,000, Abcam; #ab97023 or #ab97080) incubated for 1.5 h at room temperature. A control rat was present on all gels at the standard internal sample control (ISC). Protein bands were visualized via chemiluminescent detection (Supersignal, Pierce, Rockford, IL, USA) in a Western blot detection system (ImageQuant™ LAS 4000—GE Healthcare Life Sciences, Chalfont, UK), and quantified by densitometry using Image J analysis software version 1.52. Targeted bands were normalized to the expression of cardiac β-actin (1:1000; Cell Signaling; #4967).

4.6. Enzymatic Activity of Cardiac Glucose Metabolism

Activities of key glucose metabolism enzymes were analyzed, including HKII, LDH, and CS. Heart LV samples were homogenized 1:20 (wt/vol) in 50mM Tris-HCl, 1mM EDTA, and protease inhibitor cocktail, pH 7.4 using a Polytron instrument (Kinematica, Littau-Lucerne, Switzerland). The lysate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected. All enzyme activities were determined at 25 °C using a Spectra Max 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA), and assay buffer without sample was used as blank. HKII assay medium consisted of 75 mM Tris-HCl, 7.5mM MgCl₂, 0.8 mM EDTA, 1.5 mM KCl, 4 mM 2 mercaptoethanol, 0.4 mM NADP⁺, 2.5 mM ATP, 1 mM glucose, 1.4 units glucose-6-phosphate dehydrogenase, and 0.05% Triton X-100. Total assay volume was 165 µL, and 10 µL of homogenate was added. The assay was initiated by adding glucose [65]. LDH was assayed in a reaction mix containing 20 mM Tris, 6.0 mM pyruvate, and 5.0 mM NADH and monitored at 340 nm. CS assay medium consisted of 50 mM Tris-HCl, 1 mM EDTA, 0.2 mM DTNB, 0.1 mM acetyl-CoA, 0.5 mM oxaloacetate, and 0.05% Triton X-100. The total assay volume was 165 uL, and 10 uL of homogenate was added. The assay was initiated by adding oxaloacetate [66]. The rate of change in absorbance was monitored at 412 nm (ε = 13.6 μmol × mL⁻¹ × cm⁻¹).

4.7. 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.

4.8. 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).

5. 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.

Author Contributions

Conceptualization, P.G.S. and A.C.C.; methodology, P.G.S., L.C.d.T., G.M.M., D.F.V., G.A.F.M. and S.L.B.d.S.; validation, L.C.d.T. and A.C.C.; formal analysis, L.P.d.C. and C.R.P.; investigation, L.C.d.T., G.M.M., V.L.S. and K.O.; resources, G.M.M. and K.O.; data curation, P.G.S. and K.O.; writing—original draft preparation, P.G.S.; writing—review and editing, P.G.S., D.F.V. and A.C.C.; visualization, P.G.S. and D.F.V.; supervision, A.C.C.; project administration, P.G.S. and A.C.C.; funding acquisition, A.C.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Ethics Committee on Animals Experiments of the Botucatu Medical School, São Paulo State University, UNESP (1081/2014-CEUA).

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.

Acknowledgments

The authors are grateful to Colin Edward Knaggs for English editing.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Table

Figure 1. Bodyweight and post mortem cardiac morphology at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]). Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). (A) BW: body weight. (B) LV/TB: left ventricle corrected by tibia (TB). (C) RV/TB: right ventricle corrected by TB; (D) AT/TB: atrium corrected by TB; (E) Lung/TB: lung corrected by TB. 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).

Figure 2. Heart rate and structural echocardiographic data at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]). Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). (A) HR: heart rate; (B) LA/AO: left atrial diameter (LA) to aortic diameter (AO) ratio; (C) LVDD: left ventricle (LV) diastolic diameter; (D) LVWDT: LV posterior wall diastolic thickness; (E) RWT: LV relative wall thickness. 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).

Figure 3. Functional echocardiographic data at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]). Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). (A) EF (%): left ventricle (LV) ejection fraction in percentage. (B) Meso (%): LV shortening of mesocardium in percentage. (C) LVPW: LV posterior wall shortening velocity. (D) E wave: early diastolic mitral inflow velocity. (E) A wave: late diastolic mitral inflow velocity. (F) E/A: ratio between E wave and A wave. 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).

Figure 4. Hypoxia-inducible factor-1α (HIF-1α) protein expression evaluated by Western blot. (A) Quantification of HIF-1α. (B) Representative bands of protein. Sham: operated control; AoS: supravalvar aortic stenosis; HF: heart failure; Sham2: Sham 2 weeks; Sham6: Sham 6 weeks; Sham18: Sham 18 weeks; ShamHF: Sham HF; AoS2: AoS, 2 weeks; AoS6, 6 weeks; AoS18, 18 weeks; AoSHF, HF. ISC: Internal control of the sample; AU: arbitrary unity. 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 = 8 animals for each group).

Figure 5. Glucose and insulin data. (A) Blood glucose. (B) Serum insulin. Group abbreviations are as defined in Figure 4. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey in (A). Data expressed in median (Min and Max) and analyzed by ANOVANP and Dunn in (B). p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments; (n = 8–12 animals for each group).

Figure 6. Cardiac insulin signaling and glucose transporters 1 and 4 at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]) evaluated by Western blot. Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). Quantification of (A) pIR: phosphorylated insulin receptor, (B) Total IR: total insulin receptor, (C) pIR/Total IR: pIR/Total IR ratio, (D) pAKT: phosphorylated protein kinase B, (E) Total AKT: total protein kinase B, (F) pAKT/Total AKT: pAKT/Total AKT ratio; (G) PI3K: phosphoinositide 3-kinase, (H) GLUT4: glucose transporter 4, and (I) GLUT1: glucose transporter 1. (J) Representative bands of proteins. 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 = 8 animals for each group).

Figure 7. Myocardial glycolysis and glycogen at different moments of euthanasia (2, 6, 18 weeks [W] and heart failure [HF]) evaluated by Western blot and enzymatic activity assay. Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). Quantification of (A) HKII: hexokinase II, (B): HK: activity of hexokinase II, (C) PFKB2: phosphofruct kinase 2, (D) PK: pyruvate kinase, and (E): cardiac glycogen. (F) Representative bands of proteins. 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 = 8 animals for each group).

Figure 8. Myocardial aerobic and anaerobic metabolism at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]) evaluated by Western blot and enzymatic activity assay. Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). Quantification of (A) PDH: pyruvate dehydrogenase, (B) CS: citrate synthase activity, (C) LDH: lactate dehydrogenase, (D) LDH: LDH activity, and (E) cardiac lactate concentration. (F) Representative bands of proteins. 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 = 8 animals for each group).

Figure 9. Overview of results observed during the evolution process of cardiac remodeling from hypertrophy to heart failure. Expr.: expression. Protein abbreviations are as defined in Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8. 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.

Figure 10. Scatter plot of canonical variables in aortic stenosis groups. AoS: supravalvar aortic stenosis; HF: heart failure; • AoS2, 2 weeks; • AoS6, 6 weeks; • AoS18, 18 weeks; • AoSHF, HF; HIF-1α: factor inducible hypoxia. (A) Insulin signaling and glucose transporters 1 and 4 (total and phosphorylated insulin receptor, total and phosphorylated protein kinase B, phosphoinositide 3-kinase and glucose transporter 1 and 4 expression); p < 0.027. (B) Glycolysis (hexokinase activity, hexokinase II, phosphofruct kinase 2 and pyruvate kinase expression); p < 0.018. (C) Anaerobic pathways (lactate dehydrogenase expression and activity); p < 0.024. (D) Aerobic pathways (pyruvate dehydrogenase expression and citrate synthase activity); p < 0.957. Canonical correlations: insulin signaling and glucose transporters 1 and 4, 0.671; glycolysis, 0.625; anaerobic pathways, 0.675; aerobic pathways, 0.054. 푢1 = 1.00 (HIF-1α). Insulin signaling and glucose transporters 1 and 4: A푣1 = 0.40 pIR + 0.23 total IR + 0.56 pAKT − 0.01 total AKT + 0.54 PI3K + 0.74 GLUT1 + 0.26 GLUT4. Glycolysis: A푣1 = 0.32 HKII + 0.66 HK + 0.81 PFK2 + 0.28 PK. Anaerobic pathways: A푣1 = 0.45 LDH − 0.99 LDH activity. Aerobic pathways: A푣1 = 0.789 PDH + 0.723 LDH.

Table 1

Clinical and pathological data of heart failure (HF).

Signs of HF	Grups	Intensity and RF
Respiratory pattern change	ShamHF	(−)
	AoSHF	(+++) 100%
Ascites	ShamHF	(−)
	AoSHF	(+++) 100%
Pleural effusion	ShamHF	(−)
	AoSHF	(+++) 100%
Thrombus in LA	ShamHF	(−)
	AoSHF	(++) 20%; (+++) 80%
Hypertrophy of RV	ShamHF	(−)
	AoSHF	(++) 10%; (+++) 90%

RF: relative frequency in percentage (%); intensity of heart failure signs: (−) absent; (++) moderate; (+++) severe; LA: left atrium; RV: right ventricle; Sham_HF: operated control (n = 15); AoS_HF: AoS with severe heart failure (n = 14).

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