Moderate Overweight of Male Rats Chronically Fed with a Hyperlipidic Diet Developed Early Silent Ang-(3–4)-Sensitive Myocardial Molecular Lesions and Preserved Echographic Parameters

1. Introduction

The combination of overweight and obesity is currently considered a true pandemic that, together with malnutrition and climate changes, is part of the first significant syndemic of the 21st century [1]. Recent data from the World Health Organization [2] indicate that global adult obesity has more than doubled since 1990 and that among adolescents has quadrupled. In 2022 [3], out of a total world population of 8 billion, 2.5 billion adults over 18 were overweight; of these, 890 million were obese. Data from 2018 [4] show that mortality associated with overweight/obesity is high: around 50/1,000 adults per year in the case of overweight and around 70/1,000 adults per year in the case of obesity. It is important to emphasize that there is a marked increase in the incidence of overweight and obesity in children, adolescents, and young adults, which evolves into the same condition – generally associated with severe comorbidities – in adulthood [5,6].

Overweight and obesity result from a long period of positive energy balance and the inability to control food (and energy) intake and expenditure [7]. The composition of diets, especially "Western" ones [8], rich in fats and sugars, has been considered for more than two decades as one of the most critical factors in the development of overweight and obesity [9,10]. Diets that are often accompanied by a high salt content [11].

Among the processes and mechanisms that lead to severe comorbidities, including hypertension, coronary disease and metabolic syndrome (dyslipidemia, type 2 diabetes), the renin aldosterone system (RAAS) plays a significant role. It contributes to insulin resistance and stimulates inflammatory processes [8]. Research from our laboratory [12] has shown that overweight rats have a hyperactivated renal tissue RAAS with upregulation of Na⁺ transporting ATPases, significant interstitial fibrosis as a result of an early inflammatory process and expansion of visceral fat.

The present study aimed to investigate cardiometabolic changes in young [13] and moderately overweight hypertensive male rats chronically feeding with a high-fat and high-caloric diet [14]. Looking for possible inflammatory cellular and molecular changes and ionic transporters in the left ventricle, potentially associated with hyperactivity of the local cardiac RAAS [15] arm coupled to type 1 Ang II receptors. Furthermore, we intended to investigate whether these changes are reflected in the echocardiographic structure and function of the heart, as well as the result of activation of the RAAS arm coupled to type 2 Ang II receptors by Ang-(3–4) [16], the antagonist of the effects of Ang II in different physiological and pathological conditions [17,18].

It is concluded that moderate overweight resulting from the chronic intake of a high-fat diet that mimics the so-called "Western diets" [8] causes early cardiometabolic changes and in the active transport of Na⁺ and Ca²⁺ in the myocardial tissue of young hypertensive rats. These changes coexist with a shortening fraction and an ejection fraction still preserved.

2. Results

2.1. Development of Moderate Overweight in Rats that Received the High-fat Diet

Figure 1 shows that rats weighing an average of 259 g at 58 days of age fed the HL diet developed moderate – but significant – overweight 106 days later compared to CTR rats. This last group, fed with a diet prepared following the recommendations of AI-93 [19] containing 385 kcal/100 g of dry food with 9 % of calories coming from lipids, reached an average body mass of 458 ± 15 g. Meanwhile, the group exposed to the hypercaloric and high-fat HL diet (574 kcal/100 g of dry food with 70 % of calories coming from lipids) reached a body mass of 506 ± 16 g, i.e. 10 % greater. These values ​​are close to the asymptotic value of the previously described growth curves [12], that is, the upper limits of growth.

It is clear that the decrease in food and energy intake in HL rats, without influence on CTR rats [12], does not explain the small changes in body mass observed in Figure 2, which could even accompany changes in other parameters on the influence of Ang-(3–4) was tested. Therefore, water intake, urinary volume, and water balance were analyzed 24 h after administration of Ang-(3–4).

Figure 3 shows that the administration of Ang-(3–4) causes an increase in water intake in CTR rats (A), which is accompanied by a proportionally similar increase in urinary volume over the same period (B). These changes in parallel result in an unmodified water balance in CTR rats receiving Ang-(3–4)(C). In the case of the treated HL group, whose liquid intake is lower (A), urinary volume (B) had values ​​that also resulted in an unmodified water balance after administration of Ang-(3–4) (C). The comparison of studies on food and caloric intake [12] and water balance (the present work) allows us to conclude that housing rodents in metabolic cages alone or accompanied by the brief administration of Ang-(3–4), has an intrinsic variability whose causes remain open, as recently demonstrated [20]. That is why these still-ununderstood modifications must be considered when analyzing materials collected in these cages or Ang-(3–4)-induced modifications in other functions, processes, or organs. This point will be discussed again later.

2.2. Biomarkers of Metabolic Alterations and Inflammatory Processes in HL Overweight Rats

Just over a decade ago, the deposition of visceral fat in the perirenal subcompartment called the renal sinus was associated with severe forms of hypertension [21]. Moreover, epididymal fat is considered a marker of visceral fat [22], in which genes are found differentially expressed in obese rats due to an alimentation with a diet rich in lipids [23]. In Figure 4, it can be seen that both epididymal fat (A) and perirenal fat (B) increased by approximately 50 % in HL rats that developed moderate overweight (~10 %) after ingesting a diet rich in lipids for 104 days. The administration of 4 doses of Ang (3–4) significantly modify visceral fat levels around the 2 organs within 48 h only in HL rats.

Obesity is frequently associated with inflammation of fat tissue, especially visceral fat, and the release of pro-inflammatory cytokines [24,25,26]. The questions formulated based on the results obtained with visceral fat (Figure 4) were: (i) would there be an increase in pro-inflammatory cytokines in left ventricular microsomes? (ii) would there be Ang-(3-4) effects? The cytokines IL-6 and TNF-α were chosen because the former is known for establishing insulin resistance associated with obesity [24]. TNF-α because of its role in the genesis of fibrosis, previously observed [27]. Regarding IL-6, Figure 5 shows that its levels in left ventricular microsomes become ~40 % higher in HL rats when compared to CTR animals (A and C). These figures also show the opposite effects of Ang-(3-4): return to CTR values ​​in the HL rats and increase to those found in HL rats not treated with Ang-(3–4). Observations that allow us to preliminarily conclude that the effects of Ang-(3–4) – as in other processes [17,18] – depend on states of activation/inhibition of pathways with different targets and functions. This point will be analyzed again in the Discussion section. When analyzing the TNF-α profile in the same microsomes, an increase of ~40 % in HL rats is also found, with a return to CTR values ​​in the HL + Ang-(3-4) group and no influence in the CTR + Ang-(3-4) group (B and D).

2.3. Atypical Metabolic Syndrome in HL Overweight Rats

The results described in this section present evidence that overweight (Figure 1 and Figure 2) and hypertension in HL rats [12,27] are accompanied by an atypical metabolic syndrome. Already at the time of the onset of hypertension, i.e., 70 days of exposure to the high-fat diet [12], HL rats present a metabolic syndrome with a peculiar characteristic. Figure 6 shows that, although total cholesterol is similar in CTR and HL rats (A), and they show a marked decrease of 50 % in high-density lipoproteins (B) and a mirrored increase in low-density lipoproteins (C), total triglycerides are also reduced by just over 50 % (D).

Changes in glucose metabolism also characterize metabolic syndrome in HL rats. They present a moderate but significant increase in fasting blood glucose (Figure 7A) and glucose intolerance (Figure 7B,C), which is already predicted by changes in the lipid profile.

2.4. Unmodified Angiotensin II Receptors in Left Ventricle Microsomes from HL Rats

Figure 8A,C shows that the abundance of AT₁R in left ventricular microsomes is not modified in HL rats compared to CTR rats. Moreover, Ang-(3–4) had no effect either. In Figure 8B,D, it is observed that there were no changes in the abundance of AT₂R. These results contrast with what is found in renal proximal tubules. In the latter, there is a marked upregulation of AT₁R suppressed by Ang-(3–4), concomitantly with the downregulation of AT₂R. This contrast suggests that the primary lesion of the recently described hepatocardiorenal syndrome in overweight rats [27] would occur in the kidneys.

2.5. Overweight Rats Present Alterations in the Active Transport of Na⁺ and Ca²⁺ in Left Ventricle that Are Reverted by Ang-(3–4)

The results below show the modifications of left ventricular ATPases, primary active transporters of Na⁺ in the plasma membrane, and Ca²⁺ across the sarco-endoplasmic reticulum membrane in HL rats. The first participates in events related to electrical activity, and the second plays a central role in the contractile process. Figure 9A shows that rats that received the high-fat diet showed a marked decrease in (Na⁺+K⁺)ATPase activity, which was recovered in animals treated with Ang-(3–4). Administration of the peptide did not influence the pump from CTR rats.

The analysis of the SERCA2a activity results (Figure 10) aimed to investigate which function best described the dependence on ATP concentration ([ATP]) because of the role of the nucleotide in excitation/contraction coupling and the importance of changes in mitochondrial energetics in the genesis of heart failure [29]. For this, two simulations were carried out using the values ​​of the velocities averages for each [ATP] using two functions: a Michaelis Menten function (Equation 1) and another that considers the existence of inhibition due to excess substrate (Equation 2), one of the most common deviations from Michaelian kinetics of physiological significance [30]. In this second case, an activation phase is followed by another of progressive inhibition, complete or incomplete. The velocity equations used were as follows.

V = (V_max × [ATP])/(K_{m ATP} + [ATP])

(1)

V = (V_max × [ATP])/{(K_{m ATP} + [ATP]) × (1 + [ATP]/K_{i ATP} )}

(2)

Figure 10A shows the curves that best fit the respective means: in black CTR, in red HL, in blue CTR + Ang-(3-4), and in green HL + Ang-(3-4). The Michaelian equation 1 was the one that best adjusted to the mean points of the HL group and equation 2 to the remaining 3 groups. Five curves were generated for each group, one for each experiment, and from the values ​​obtained, V_max, K_{m ATP}, and K_{i ATP} were calculated (Figure 10B, C, and D, respectively). About V_max, Km ATP, and K_{i ATP}, no significant differences were found between the CTR and HL groups, nor when analyzing activities with [ATP] = 5 mM (t = 0.66; p = 0.5269), a concentration close to that of cardiomyocytes in both contraction and relaxation [31]. The differences in V between these 2 groups become significant at lower ATP concentrations, such as 1 mM (t = 5.65, p = 0.0005). Ang-(3 – a 4) stimulated SERCA2a activity, increasing V_max and K_{m ATP} in both the CTR and HL groups, decreasing K_{i ATP} in CTR rats, and causing the appearance of K_{i ATP} in HL rats. However, the response of HL animals concerning V_max and K_{m ATP} was lower than that of CTR animals. (Figure 10B, C, D).

2.6. There Is no Structural Remodeling in Overweight Rats at Juvenile Age

Figure 11 presents the results of echocardiographic studies designed to investigate whether there are indicators of structural remodeling of the left ventricle in moderately overweight and hypertensive rats. Measurements of the internal diameters of the left ventricle at the end of diastole and systole, as well as of the left ventricular end-diastolic and end-systolic volumes, were recorded at the level of the region where the papillary muscles are located (red lines and circles). These measurements (representative recordings in panels A, B, C, and D, respectively) demonstrate that HL rats do not present changes in the fractional shortening (Figure 11D) or the ejection fraction (Figure 11E) and that there were no effects of Ang-(3–4).

3. Discussion

The main findings of the present work are that moderate overweight (Figure 1) in young rats [13] chronically fed a high-fat and high-caloric diet [14] (HL) present – in addition to the gradually installed arterial hypertension – changes in central areas that regulate thirst [32] since liquid intake is markedly lower and, consequently, urinary excretion (Figure 3). A marked and puzzling decrease in plasma triglyceride levels appears to be associated with the non-alcoholic steatosis these rats present [27]. Evidence of the role of upregulation of the RAAS axis coupled to Ang II type 1 receptors (AT₁R) in the left ventricle arises from the observation that increased levels of the pro-inflammatory cytokines IL-6 and TNF-α return to normal values ​​by administering Ang-(3–4) to upregulate signaling coupled to type 2 Ang II receptors (AT₂R). The participation of RAAS also emerges from the observation that, in the myocardium of HL rats, the decrease in (Na⁺+K⁺)ATPase and the increase in the ouabain-resistant Na⁺-ATPase are also normalized by Ang-(3–4), as well as the dependence on ATP concentration characterized by inhibition by excess substrate of the sarco-endoplasmic reticulum Ca²⁺-ATPase, which was lost in HL rats. However, the important metabolic alterations and notable changes in ion transport ATPases from overweight rats still coexist with fractional shortening and ejection fraction preservation.

Although moderate, the overweight of HL rats is accompanied by a significant increase in visceral fat around the epididymis and in the perirenal region, an observation accompanied by evidence of hyperactivity of the local RAAS coupled with AT₁R. Supporting this idea is the observation that the acute administration of just 4 doses of Ang-(3-4) in the short space of 48 h to antagonize AT₂R-coupled signaling partially reverses the expansion of these biomarkers of increased visceral fat in HL rats, without any effect in CTR rats (Figure 4). This selective sensitivity indicates the existence of a pro-hypertensive tissue microenvironment [12,17] with upregulation of the Ang II → AT₁R axis. In light of this set of findings, it could be proposed − in terms of mechanisms − that the abnormal activation of this axis would promote lipogenesis in HL rats [33,34,35], while the allosteric activation [16] of the antagonist Ang II → AT₂R pathway would restore lipase-mediated catalysis in adipocytes [34,35,36].

The existence of an intense inflammatory state – stimulated by the Ang II → AT₁R pathway – that reaches the heart is confirmed by observations related to the levels of pro-inflammatory cytokines in the left ventricular tissue (Figure 5), which doubled in HL rats, being normalized by Ang-(3–4). We chose to investigate IL-6 due to its known effect on establishing insulin resistance associated with obesity [24] and because it is considered a predictor of mortality in cardiorenal syndromes [37]. The upregulation of TNF-α levels in the left ventricle, in turn, reveals the existence of a link between a process of local inflammation caused by Ang II and subsequent fibrosis due to the differentiation of monocytes into fibroblasts [38], in route to organ remodeling with congestive heart failure [39]. In this context, the significant increase in IL-6 in CTR rats caused by Ang-(3–4) deserves special discussion in the context of the dual effects of this cytokine [40,41]. In normonourished animals Ang-(3–4) would increase the release of the cytokine to exert anti-inflammatory effects, promoting the alternative activation of macrophages to the M2 phenotype [42]. This activation would occur in tissue conditions where intracellular mediators favor IL-6 trans-signaling [41,43], e.g. a physiological status of the local RAAS [44].

A metabolic syndrome associated with moderate overweight in HL rats is evidenced by the lipidogram and blood glucose profiles indicating insulin resistance (Figure 6 and Figure 7). The biochemical remodeling of plasma lipoproteins with a decrease in HDL and an increase in LDL is classically described for the dyslipidemias of metabolic syndrome, representing a global metabolic effect of insulin resistance [45,46], which correlates with the increase in visceral fat described above and with the changes in fatty acid fluxes and lipid deposits in the liver found in the hepatocardiorenal syndrome of HL rats [27]. The maintenance of total cholesterol in the HL group at CTR levels would probably results, in turn, from the combination of changes in lipid metabolism in different tissues that compensate for each other and reverberate in the plasma [47]. The marked and atypical decrease in plasma triglycerides could also be linked to the pronounced hepatic steatosis that ultrasound reveals in HL rats [27].

The components of metabolic syndrome with insulin resistance in HL rats, which has the augmented production of cytokines by the visceral fat as a central physiopathogenic factor [48,49], are completed with fasting hyperglycemia and glucose intolerance, completing a network of processes and mechanisms that associate visceral adiposity, insulin resistance, hypertension, changes in body Na⁺, and cardiovascular risk [12,50]. Although fasting hyperglycemia and insulin resistance are mild in the HL group, it should be noted that these are young rats. Juvenile obesity, in addition to leading to a progressive worsening of insulin resistance over time, significantly increases cardiovascular risk in adulthood, as demonstrated by the evaluation and comparison of different circulatory parameters in humans [51].

The inflammatory state of the myocardial tissue that the cytokines revealed and the incipient fibrosis process [27] indicate RAAS hyperactivity. This hyperactivity would result from the selective increase/decrease of targets in steps after the binding of Ang II to AT₁R (and a decrease in signaling coupled to AT₂R), because the abundance of both receptors in the left ventricle microsomal fraction remained similar in the 4 groups as shown in Figure 8. Well-known targets downstream of Ang II binding to AT₁R are MAP kinases (ERK1/2, JNK, p38, MAPK), NADPH oxidase (which would lead to increased production of reactive O₂ species) [52,53], PKC (upregulated) and PKA (downregulated) [54,55]. This ensemble, associated with various processes, can be expanded by considering protein kinases involved in cardiac inflammation and remodeling. These altered Ang II → AT₁R and/or Ang II → AT₂R signaling could likely be present in HL rats.

The impact of overweight on Na⁺-transporting ATPases (Figure 9) and SERCA2a (Figure 10) also constitutes a central element in the pathophysiology of cardiac structural and functional changes that can occur in HL rats. Na⁺-transporting ATPases because they participate in events related to electrical activity and are involved in dysfunctions described in cardiometabolic diseases [56]. Downregulation of (Na⁺+K⁺)ATPase potentiates apoptosis in cardiomyocytes [57] and oxidative damage [58], which would be especially critical when ROS production is increased by activation of the Ang II → AT₁R axis [59]. Regarding the ouabain-resistant Na⁺-ATPase, its functioning in normonourished rats contributes to normal electrical activity [54]. The upregulation of this Na⁺ pump due to RAAS hyperactivity in overweight rats could lead to changes in the regulation of cardiomyocyte volume [60] and, therefore, in the function of these cells as a whole [61].

The relevance of potential changes in SERCA2a in HL rats emerges from the fact that its changes impact the contractile process in heart failure [62]. As seen in Figure 10, there are no differences in SERCA2a activity at 5 mM ATP, a concentration close to that encountered in cardiomyocytes in both contraction and relaxation [31]. Mitochondria replenish cytosolic ATP at a rate appropriate to its consumption during contraction [63,64]. The fact that there is a difference in low concentrations of ATP makes it evident that if myocardial inflammatory damage [65] in overweight is accentuated to the point of compromising mitochondrial function [66], the activity of SERCA2a will also be affected. A SERCA2a dysfunction that leads to decreased activity would accelerate the progression to heart failure [67,68] in HL rats. Concerning the stimulation by Ang-(3–4), the fact that the response in HL rats was smaller than in CTR rats suggests that chronic administration of the high-fat diet affected the ability of the Ang II → AT₂R axis to counter-regulate the Ang II → AT₁R axis and therefore some of the changes resulting from the activation of the latter. At this point, it is important to discuss the recovery of the kinetics of SERCA2a inhibition by excess substrate, one of the finest mechanisms of key enzyme activity [69,70]. Inhibition of SERCA2a activity by high ATP makes it sensitive to energy demand during systolic sarcomere contraction during [71]. Thus, its loss in HL rats, recovered by the administration of Ang-(3–4), again indicates a pathological effect at the molecular level resulting from the upregulation of the Ang II → AT₁R axis of the RAAS, which is counterbalanced by the activation of the Ang II → AT₂R axis. In terms of injury mechanisms, it could be that high tissue levels of ROS found in overweight/obesity [8] would be responsible for structural and functional changes in SERCA2a due to the oxidation of cysteines and the nitration of tyrosines [72], which are key amino acids in the enzyme catalysis domains [73].

Although the relationship between obesity and heart failure has been the subject of an increasing number of studies in recent years (for an illustrative review, see [74]), two questions have not yet been answered. They are: (1) Can moderate overweight be associated with or precede early heart failure? (2) What are the effects of inflammation and changes in ion transport in myocardial tissue associated with overweight/obesity? Cardiometabolic diseases in obesity, in which there is an important component of generalized inflammation, evolve and worsen when there is hypertension [75,76]. HL rats became progressively hypertensive throughout the exclusive administration of the high-fat diet [12] and presented myocardial fibrosis [27] despite being moderately overweight. This ensemble of observations adds to the changes in renal Ang II receptors already described [12]. They allow us to propose that the triad known as cardiovascular-kidney-metabolic syndrome [77] should even include moderate weight gains, which, as in the case of HL rats, already have a significant expansion of visceral adiposity. In this initial stage of the evolution of fat mass and cellular and molecular changes in the myocardium, including marked inflammation and important changes in ionic transporters involved in excitation/contraction coupling, there is still no evidence of systolic dysfunction. As shown in Figure 11, both shortening fraction and ejection fraction, which are central measures of left ventricular systolic function [78,79,80], are preserved. Ang-(3–4) was also without effect, probably because the downstream upregulation of the Ang II → AT₁R axis, discussed above, has not yet reached the macroscopic structural remodeling that echocardiography can reveal.

4. Materials and Methods

4.1. Ethical Considerations

All experimental procedures were approved by the Ethics Committee for Using Animals in Research at the Federal University of Rio de Janeiro under protocol number 075.19. The execution of these procedures adhered to the Committee's Guidelines, which follow the Uniform Requirements for Manuscripts Submitted to Biomedical Journals established by the International Committee of Medical Journal Editors and the ARRIVE guidelines [81].

4.2. Experimental Groups and Diets

At birth, the rats were distributed so that each mother remained with 8 animals, ensuring that all had equal nutritional access [82]. All male animals were preserved, and, if necessary, the maximum number of offspring per mother was supplemented with females. The animals were weaned on day 21 of life, started to receive a commercial diet for rodents (Neovia Animal Nutrition, Contagem, Brazil), and were regrouped to avoid the litter effect [83] in cages containing 5 animals until the 57 days of life in a controlled environment at 23 ± 2 ^oC with a 12 h/12 ​​h light and dark cycle.

Upon reaching 58 days of life, the animals were randomly separated into 2 groups. The control (CTR), with free access to a diet containing 36, 292, and 57 kcal/100 g dry weight chow from lipids, carbohydrates, and proteins, respectively, totaling 385 kcal/100 g. The high lipid (HL) group received 402, 103, and 69 kcal/100 g from lipids, carbohydrates, and proteins, respectively [14]. The Na⁺ content, measured by flame photometry, varied between 5.1–5.8 and 7.7–8.0 mequiv/100 g dry chow (CTR and HL, respectively) [12]. For greater detail on the diets, see Supplement Table S1 in [12]. Both groups had free access to filtered water. Both CTR and chow HL food were purchased from PragSoluções (Jaú, Brazil). Upon reaching 104 days of diet (162 days of age), the rats were separated into 2 more groups – also randomly – and housed individually in metabolic cages so that half received by gavage 4 doses (80 mg/kg body mass diluted in water) of Ang-(3–4) (AminoTech, Sorocaba, Brazil). Untreated rats received only the vehicle.

After 48 days of treatment, the rats were sacrificed by decapitation. Each animal's heart, perirenal, and epididymal fat were immediately collected using surgical scissors. The organs were dried with filter paper to remove excess blood and weighed. Perirenal and epididymal fat masses were considered markers of visceral fat [22], as described above.

4.3. Lipidogram

After 70 days of consumption of the CTR or HL diets (128 days of life), the rats were fasted for 10 h (water ad libitum) and anesthetized after this period with ethyl ether. Blood was collected by caudal puncture, centrifuged at 13,000× g for 5 min, and plasma was collected to measure total cholesterol/TC, HDL cholesterol, and triglycerides/TG. Commercial kits (Bioclin, Belo Horizonte, Brazil) were used: Bioclin K083 mono reagent total cholesterol, Bioclin enzymatic HDL cholesterol, and Bioclin K117 mono reagent triglycerides.

4.4. Glycemia Determination and Oral Glucose Tolerance Curve

After 95 days of diet (153 days of age), some of the animals that received the CTR or HL diets were fasted for 10 h (water ad libitum) to measure blood glucose. Immediately afterward, they received 2 g of glucose/kg body mass by gavage. The blood glucose measurement was done using the touch-ultra mini glucometer (OneTouch^®-LifeScan, Milpitas, CA, USA), taking the fasting blood glucose value to construct the curve (time zero). The measurements were taken 15, 30, 50, 60, and 120 min apart.

4.5. Preparation of Left Ventricle Microsomes

After sacrifice (at 106 days of exposure to CTR or HL diets, 164 days of life), the heart of each animal was immersed in a solution containing 250 mM sucrose, 1 mM imidazole, 1 mM EDTA, and 0.15 mg/mL of trypsin inhibitor (pH adjusted to 7.6 with TRIS) [84]. Each heart was carefully dissected to obtain the left ventricle, weighed, and cut into smaller fragments with the aid of surgical scissors. A pool of 2–3 ventricles was suspended in the same solution (1 g of tissue in 4 mL).

The suspension was mechanically homogenized at 4 ^oC using a Potter Elvejhem homogenizer with a Teflon pestle (5 cycles of 1 min at 1,700 rpm with 10 s pauses). Homogenates were sequentially centrifuged twice (1,650× g for 15 min at 4 ^oC, 70 Ti Beckman rotor) to remove debris and nuclei and then the supernatant was centrifuged at 115,000× g for 60 min. The resulting precipitate was recovered and suspended in 250 mM sucrose solution and stored in liquid N₂. Protein concentration was determined by the Lowry method [85]. This microsomal fraction has a sarco-endoplasmic reticulum content of 8–10%, which allows simultaneous assessment of (Na⁺+K⁺)ATPase, ouabain-resistant Na⁺-ATPase, and sarco-endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) activities.

4.6. SDS PAGE and Western Blotting

Western blotting evaluated the abundance of (Na⁺+K⁺)ATPase, Ang II receptors, IL-6, and TNFα. Microsome samples (80 μg of protein) were loaded onto a gel containing 10% polyacrylamide and SDS [86]. After the separation and transfer to nitrocellulose membranes (GE Healthcare, Chicago, IL, USA), the membranes were incubated for 1 h in a solution containing 5% skimmed milk powder diluted in Tris-buffered saline (pH 7.6). The membranes were incubated overnight with the corresponding primary antibody, remaining exposed after 1 h, and incubated with the secondary antibody for subsequent development.

The primary antibodies were as follows. Against the α-catalytic subunit of (Na⁺+K⁺)ATPase, the catalog antibody A276 (Sigma-Aldrich, Saint Louis, MO, USA) was used at a dilution of 1:1,000. For the cytokines IL-6 and TNFα, antibodies from Imuny Biotechnology (Campinas, Brazil; catalog IM-0407) at a dilution of 1:1,000 and from Immuny Biotechnology (catalog IM-406) at a dilution of 1:1,000 were used, respectively. For AT₁R, the anti-rabbit polyclonal antibody was used (Alomone Labs, Jerusalem, Israel; catalog AAR-011, at a dilution of 1:500). For AT₂R, the anti-rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA; catalog SC-9040, dilution 1:250). The secondary antibodies were anti-mouse and anti-rabbit from GE Healthcare (NA934 and NA931, respectively), both at 1:5,000 dilution. The loading control was performed through immunodetection of GAPDH (monoclonal antibody SC-32233, Santa Cruz Biotechnology). The LAS 4000 system equipment (GE Healthcare) and the ImageJ software (NIH) were used to detect the immunosignals.

4.7. Measurement of Left Ventricle Na⁺-transporting ATPases Activity

The activity of the two Na⁺-transporting ATPases was assessed by measuring the orthophosphate released during ATP hydrolysis [87]. For the (Na⁺+K⁺)ATPase experiments, microsome samples (0.05 mg/mL) were preincubated at 37 ^oC for 10 min in a solution containing 50 mM Bis-TRIS propane (pH 7.4), 0.2 mM EDTA, 120 mM NaCl, and 5 mM MgCl₂, in the absence or presence of 5 mM ouabain. The reaction was started by adding a solution containing 5 mM ATP and 24 mM KCl (final concentrations) and terminated 10 min later by adding a suspension of activated charcoal in 1 N HCl (1 vol:1 vol). For the subsequent colorimetric reaction, the reaction tubes were centrifuged at 13,000x g for 15 min to remove charcoal. Next, the supernatant was removed and mixed with ferrous sulfate-molybdate solution (1 vol:1 vol). (Na⁺+K⁺)ATPase activity was calculated by subtracting the activity obtained in the presence of ouabain from the total activity.

The ouabain-resistant and furosemide-sensitive Na⁺-ATPase activity was assessed by first preincubating microsome samples (0.2 mg/mL) in a solution containing 20 mM Hepes-TRIS (pH 7.0), 10 mM MgCl₂, 120 mM NaCl, and 2 mM ouabain, in the presence or absence of 2 mM furosemide. The reaction (at 37 ^oC) was started by adding 5 mM ATP and finished 10 min later, following the process described for the (Na⁺+K⁺)ATPase. The activity of the ouabain-resistant and furosemide-sensitive Na⁺-ATPase was determined by the difference between the activities measured in the absence and presence of furosemide.

4.8. Measurement of Left Ventricle Sarco-Endoplasmic Reticulum Ca²⁺-ATPase/SERCA2a Activity

SERCA2a activity was assayed at increasing ATP concentrations (0.1, 0.2, 0.5, 1, 3 and 5 mM). Microsomes (0.05 mg/mL) were initially preincubated at room temperature for 20 min in the absence or presence of 4.6 μM thapsigargin, in a solution containing 50 mM TRIS-HCl (pH 7.0), 160 mM sucrose, 10 mM NaN₃, 0.2 mM ouabain, 1.0 mM EGTA and sufficient MgCl₂ and CaCl₂ concentrations [88] to obtain free Mg²⁺ and Ca²⁺ concentrations of 0.5 mM and 20 μM, respectively, for each ATP concentration. After preincubation, ATP was added to obtain the final concentrations mentioned above. After incubation for 15 min at 37 ^oC, the reaction was stopped by adding the charcoal suspension in acid and the samples processed as in the case of Na⁺-transporting ATPases. SERCA2a activity was calculated as the difference between total activity and that obtained in the presence of thapsigargin.

4.9. Echocardiographic Images

Echocardiography studies were performed to evaluate cardiac structure/function in vivo. The studies were conducted using a Vevo 2100 High-Resolution Imaging System (VisualSonics, Toronto, Canada) coupled to a 21 MHz transductor. Rats (aged 164 days; 106 days of exposure to the different diets, including the two days under Ang-(3–4 treatment) were maintained under isoflurane anesthesia during the study. Images were acquired in bidimensional and M mode [89] and then analyzed by a blinded specialist using the VevoLab software.

4.10. Statistical Analysis

Results, which were analyzed using GraphPad Prism 8.0.2, are presented as mean ± SEM. Two means were compared using the paired or unpaired Student's t-test, as described in the captions of each figure. For comparisons between more than two means, 2-way ANOVA was used. Differences were considered significant at p < 0.05.

5. Conclusions

In conclusion, our research demonstrates that being very moderately overweight already leads to critical processes of molecular damage in myocardial tissue, which are revealed by inflammatory biomarkers. These accentuated molecular lesions can alter the left ventricle's hemodynamic function, which is still preserved. Future directions of the study point to the challenge of characterizing altered components of signaling pathways downstream of the cardiac Ang II receptors, whose abundance remains unmodified in moderate overweight. At this initial pathological stage, it will be central to detect intervention targets to prevent progression to systolic dysfunction and cardiac remodeling.