All experiments and procedures were performed in accordance with the National Institutes of Health’s (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee for Animal Experimentation of the Botucatu Medical School, UNESP, São Paulo, Brazil (1126/2015). A total of 242 male Wistar rats weighing 200 to 250 g were used in this study. MI was induced by coronary artery ligation, as previously described.^{22 , 23}

After surgery, the animals were placed in boxes with six animals each. Seven days after surgical procedure, the first echocardiographic study was performed to evaluate the efficacy of the surgical procedure.²⁴ Then, the animals were randomly placed in boxes with two animals each, to receive either OJ or a maltodextrin (M) solution. The groups were 1) SM, sham animals that received M solution (n = 20); 2) SOJ, sham animals that received OJ (n = 20); 3) IM, infarcted animals that received M solution intake (n = 40); and 4) IOJ, infarcted animals that received OJ (n = 40). The sample size used was based on other studies from our laboratory.^{3 , 8 , 25} The number of rats in the infarcted groups was higher, since the expected mortality in these animals during the experimental period is around 50%. In addition, only rats with a left ventricular (LV) infarcted area greater than 30% were included.²⁴

Food was supplied ad libitum . The animals were treated for 3 months, and mortality was observed in this period ( Figure 1 of the supplementary data). The rats were housed in a temperature-controlled room (22 ± 2°C) with a 12-h light/12-h dark cycle.

Figure 1

MI was conducted by coronary artery ligation, as previously described.^{22 , 23} In brief, the rats were anesthetized with ketamine (70 mg/Kg) and xylazine (1 mg/Kg), and after a left thoracotomy, the heart was exteriorized. The left atrium was retracted to facilitate the ligation of the left coronary artery with 5-0 mononylon between the pulmonary outflow tract and the left atrium. The heart was then replaced in the thorax, the lungs were inflated by positive pressure, and the thoracotomy was closed. A sham group, in which animals were submitted to surgery but without coronary occlusion, was also created. After anesthetic effect, the rats were medicated orally with metamizole sodium (30 mg / kg Dipirona®, Biovet, Vargem Grande Paulista, Sao Paulo, Brazil).

Supplemented groups (SOJ and IOJ) received OJ ad libitum . Control groups (SM and IM) received a solution of water and M at a concentration of 100 g/L. The M solution was given to control animals to provide the same amount of carbohydrates as the OJ. Treatment began seven days after surgery. The OJ and M solutions were changed every 24 hours, and intake was monitored daily. Nutritional composition of the OJ is shown in supplementary data.

After three months, all rats were weighed and evaluated by transthoracic echocardiography.^{26 , 27} For the echocardiographic study, the rats were anaesthetized with intramuscular injection of ketamine (50 mg/kg) and xylazine (1 mg/kg) solution. All measurements were made by the same observer, according to the leading-edge method recommended by the American Society of Echocardiography/European Association of Echocardiography.²⁸ Echocardiography was performed with the General Electric Vivid S6 System (GE Medical Systems, Tirat Carmel, Israel) equipped with a 5- to 12-MHz phased array transducer.

After echocardiography, the animals were euthanized with a large dose of pentobarbital, and their hearts were removed. The left ventricle was isolated and LV samples were immediately frozen and stored at –80°C. One transverse section of the LV was separated and fixed in 10% buffered formalin and was then embedded in paraffin for histological study.

Five-micrometer-thick sections were stained with hematoxylin and eosin for calculations of infarction size as previously described. All animals were included in the morphometric analysis. After infarction size calculation, infarcted animals with less than 30% of LV infarcted area were excluded from analysis. All images were collected with a video camera attached to Leica microscope; the images were analyzed with the Image-Pro Plus 3.0 software program (Media Cybernetics, Silver Spring, MD).

LV samples (100 mg) were used to determine total protein and lipid hydroperoxide (LH) concentrations, and activity of the following antioxidant enzymes – GPx (E.C.1.11.1.9), superoxide dismutase (SOD, E.C.1.15.1.1), and catalase (E.C.1.11.1.6). Cardiac energy metabolism was assessed by 3-hydroxyacyl coenzyme-A dehydrogenase (OHADH; E.C.1.1.1.35.), phosphofructokinase (PFK; E.C.2.7.1.11), lactate dehydrogenase (LDH; E.C.1.1.1.27), pyruvate dehydrogenase (E.C.1.2.4.1), citrate synthase (CS; E.C.4.1.3.7.), and adenosine triphosphate (ATP) synthase (EC 3.6.3.14) activities.^{3 , 9} The enzyme activity assays were performed at 25°C with a microplate reader (µQuant-MQX 200-EONC with Gen5 2.0 software connected to a computer system control; Bio-Tec Instruments, VT, USA). All the reagents were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA).

Interferon-γ (IFN-γ) and interleukin-10 (IL-10) concentrations in LV samples were determined by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Western blot was performed to analyze protein expression of GPx-1 (ab 22604 - Abcam Inc, Cambridge), HO-1 (ab13248 - Abcam Inc, Cambridge), total and phosphorylated NF-kB (NF-kB- sc 8008 and sc 3302- Santa Cruz Biotechnology, Inc, Europe), and sirtuin-1 (Sirt-1- sc 15404-Santa Cruz Biotechnology, Inc, Europe), in total cellular extract. To determine nuclear erythroid factor 2 (Nrf-2-sc 722-Santa Cruz Biotechnology Inc, Europe), LV samples were extracted with nuclear extraction buffer.⁹ Samples were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel, and the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk and then incubated with primary and secondary antibodies. Glyceraldehyde-3-phosphate dehydrogenase GAPDH (sc 32233, Santa Cruz Biotechnology, Inc., Europe) was used for normalization of all proteins.

The normality of the data was verified by Kolmogorov–Smirnov statistical test. Data are presented as the mean ± standard deviation (SD). Variables with normal distributions were analyzed by 2-factor analysis of variance, which gives three p values: 1) factor 1, presence of MI (I); 2) factor 2, OJ intake (OJ); and 3) interaction between factors I and OJ. The 2-factor analysis of variance requires an assumption of normality. If a measurement variable does not fit a normal distribution, data transformation was performed. The Student’s unpaired t -test was used to analyze the initial echocardiogram; the χ²test was used to evaluate mortality, and Student’s unpaired t-test used to evaluate the infarct size in infarcted animals. Differences were considered statistically significant if p <0.05. Statistical analyses were performed using SigmaPlot for Windows 12.0 (Systat Software Inc., San Jose, CA).

MI led to adverse CR. Regarding morphological data, MI led to higher values of LV diastolic diameter/BW, LV systolic diameter/BW, left atrial diameter/aorta, LV mass index (LVMI), LV weight/BW, and right ventricular weight/BW ( Table 1 ), LV posterior wall thickness/BW, interventricular septum wall thickness/BW, and left atrial diameter/BW ( Table 1 of Supplementary material). These changes characterize the enlargement of the left cavities and LV hypertrophy. MI impaired cardiac systolic function, as shown by the lower values of fractional area change (FAC), S´ mean ( Table 1 ), endocardial fractional shortening, and ejection fraction ( Table 2 of Supplementary material). Cardiac diastolic function was also impaired, as shown by decreased mean E´ ( Table 1 ), E wave deceleration time, E´ lateral, and E´ septal ( Table 2 of Supplementary material) and increased A wave, A´ mean; E/E´ ratio ( Table 1 ), Tei index, E/A ratio, isovolumetric relaxation time adjusted by heart rate, A´ lateral, and A´ septal.

Table 2

Variable	SM (n = 8)	SOJ (n = 8)	IM (n = 4)	IOJ (n = 4)	p (I)	p (OJ)	p (I×OJ)
LH (nmol/g)	209±28.3	215±28.3	256±28.0	277±26.0	<0.001	0.293	0.573
Catalase (µmol/g)	64.4±6.79	61.8±7.07	55.4±7.00	58.1±7.60	0.056	0.993	0.404
SOD (nmol/mg)	12.9±2.83	13.5±2.83	18.7±1.80	17.1±1.40	<0.001	0.639	0.355
GSH-px activity (nmol/mg)	62.5±6.51	50.2±7.07	58.0±7.00	45.3±6.00	0.134	<0.001	0.951
GSH-px expression (arbitrary unit)	7.56±7.39	7.62±10.8	6.63±5.82	6.37±3.78	0.600	0.649	0.546
IL-10 (pg/mg)	23.9±7.92	39.6±13.0b	42.0±9.60A	34.1±7.60	0.135	0.352	0.008
IFN-γ (pg/mg)	7.1±2.26	13.2±3.96b	17.7±6.80A	11.2±2.00a	0.037	0.928	0.003
NF-κB (arbitrary unit)	7.03±11.0	4.61±4.98	3.40±3.12	6.80±4.00	0.942	0.330	0.266
p NF-κB (arbitrary unit)	5.40±10.2	3.18±2.40	3.28±3.12	5.22±3.34	0.587	0.084	0.980
Sirt 1 (arbitrary unit)	1.81±0.82	2.34±1.41	2.42±1.44	1.65±0.56	0.925	0.813	0.203

Data are expressed as mean ± standard deviation. n: numbers of animals included in each experimental group. SM: sham animals that received maltodextrin; SOJ: sham animals that received orange juice; IM: infarcted animals that received maltodextrin; IOJ: infarcted animals that received orange juice. LH: lipid hydroperoxide; SOD: superoxide dismutase; GSH-px: glutathione peroxidase; IL-10: interleukin-10; INF-γ: interferon- γ; NF-κB: nuclear factor–κB total and phosphorylated (p NF-κB); Sirt 1: sirtuin 1. pI: p value for the effect of infarction. pOJ: p value of orange juice intake effect. pIxOJ: p value of interaction.Bold numbers represent statistically significant effects. ^a : IM≠IOJ; ^b : SM≠SOJ; ^A : SM≠IM e ^B: SOJ≠IOJ.

MI also increased oxidative stress, as presented with higher LH and SOD activity ( Table 2 ), lower expression of HO-1 (Figure 1A), and of Nrf2 (Figure 1B). The inflammatory mediators IL-10 and INF-γ were higher in MI ( Table 2 ), and there was no difference in NF-κB and Sirt-1 between infarcted and noninfarcted animals ( Table 2 and Figure 2 of Supplementary material). A greater oxidation of carbohydrates than fatty acids and impaired energy metabolism were observed, as shown by higher activity of LDH and PFK and lower activity of OHADH, CS, and ATP synthase. No difference was observed for activity of pyruvate dehydrogenase ( Table 3 ).

Figure 2

Table 3

Variable	SM (n = 8)	SOJ (n = 8)	IM (n = 4)	IOJ (n = 4)	p (I)	p (OJ)	p (I×OJ)
PFK (nmol/g)	128±17.0	112±24.6	139±26.0	178±26.0aB	<0.001	0.257	0.011
LDH (nmol/mg)	88.5±18.7	82.6±18.1	111±18.6	134±16.	<0.001	0.320	0.092
PDH (nmol/g)	344±53.7	385±31.1	345±100	341±42.0	0.386	0.461	0.376
OHADH (nmol/mg)	33.2±6.22	33.9±6.22	23.8±8.40	24.5±2.80	0.003	0.798	0.992
CS (nmol/mg)	50.0±6.22	49.4±6.51	34.5±7.40	40.5±4.80	<0.001	0.350	0.254
ATP synthase (nmol/mg)	21.0±3.11	27.8±5.37	11.4±1.20	15.9±4.40	<0.001	0.005	0.532

Data are expressed as mean ± standard deviation. n: numbers of animals included in each experimental group. SM: sham animals that received maltodextrin; SOJ: sham animals that received orange juice; IM: infarcted animals that received maltodextrin; IOJ: infarcted animals that received orange juice. PFK: phosphofructokinase; LDH: lactate dehydrogenase; PDH: pyruvate dehydrogenase; OHADH: 3-hydroxyacyl coenzyme-A dehydrogenase; CS: citrate synthase; ATP: adenosine triphosphate. pI: p value for the effect of infarction. pOJ: p value of orange juice intake effect. pIxOJ: p value of interaction. Bold numbers represent statistically significant effects.^a: IM≠IOJ;^b: SM≠SOJ;^A: SM≠IM e^B:SOJ≠IOJ.

OJ intake reduced the LV cavity, with lower values of LV end-diastolic diameter (LVDD) and LV end-systolic diameter (LVSD); improved systolic function, with higher values of S´ mean; and improved diastolic function, with lower left atrial diameter adjusted for aorta diameter ( Table 1 )²⁹ after MI. No differences were observed for other echocardiographic variables ( Table 1 ). The other function variables were not valued because of the higher heart rate³⁰ in the IOJ group (presented in Supplementary material Table 2 ).

In addition, the animals that consumed OJ presented lower activity of GSH-Px ( Table 2 ). No differences were observed for LH, SOD activity, catalase activity, or GSH-Px expression ( Table 2 and Figure 2 of the Supplementary material).

Regarding the inflammatory mediators, the SOJ group showed higher IL-10 and INF-γ than the SM group ( Table 2 ). Also, the IOJ group had lower INF-γ values than the IM group ( Table 2 ). No differences were observed for NF-κB or Sirt-1 between animals that consumed or did not consume OJ ( Table 2 and Figure 2 of the Supplementary material).

An improvement in the use of substrate occurred in the animals that consumed OJ. We observed higher values in PFK activity in the IOJ group compared with the IM group and higher activity of ATP synthase in animals that consumed OJ. No difference was observed in the activity of other energy metabolism enzymes between the groups ( Table 3 ).

Interestingly, the animals with OJ intake had a higher expression of HO-1 (Figure 1A), although they did not present a difference in Nrf2 expression (Figure 1B).

The OJ used in the study was a commercial, ready-to-eat, pasteurized juice, free of preservatives and sugar. The choice of ready juice was to ensure its standardization. However, the use of other types of juices, made with other types of oranges, could possibly show different responses.