Effects of concurrent training and N-acetylcysteine supplementation on cardiac remodeling and oxidative stress in middle-aged spontaneously hypertensive rats

Abstract

Background

This study evaluated the effects of concurrent isolated training (T) or training combined with the antioxidant N-acetylcysteine (NAC) on cardiac remodeling and oxidative stress in spontaneously hypertensive rats (SHR).

Methods

Six-month-old male SHR were divided into sedentary (S, n = 12), concurrent training (T, n = 13), sedentary supplemented with NAC (SNAC, n = 13), and concurrent training with NAC supplementation (TNAC, n = 14) groups. T and TNAC rats were trained three times a week on a treadmill and ladder; NAC supplemented groups received 120 mg/kg/day NAC in rat chow for eight weeks. Myocardial antioxidant enzyme activity and lipid hydroperoxide concentration were assessed by spectrophotometry. Gene expression of NADPH oxidase subunits Nox2, Nox4, p22 phox, and p47 phox was evaluated by real time RT-PCR. Statistical analysis was performed using ANOVA and Bonferroni or Kruskal–Wallis and Dunn.

Results

Echocardiogram showed concentric remodeling in TNAC, characterized by increased relative wall thickness (S 0.40 ± 0.04; T 0.39 ± 0.03; SNAC 0.40 ± 0.04; TNAC 0.43 ± 0.04 *; * p < 0.05 vs T and SNAC) and diastolic posterior wall thickness (S 1.50 ± 0.12; T 1.52 ± 0.10; SNAC 1.56 ± 0.12; TNAC 1.62 ± 0.14 * mm; * p < 0.05 vs T), with improved contractile function (posterior wall shortening velocity: S 39.4 ± 5.01; T 36.4 ± 2.96; SNAC 39.7 ± 3.44; TNAC 41.6 ± 3.57 * mm/s; * p < 0.05 vs T). Myocardial lipid hydroperoxide concentration was lower in NAC treated groups (S 210 ± 48; T 182 ± 43; SNAC 159 ± 33 *; TNAC 110 ± 23 *^# nmol/g tissue; * p < 0.05 vs S, ^# p < 0.05 vs T and SNAC). Nox 2 and p22 phox expression was higher and p47 phox lower in T than S [S 1.37 (0.66–1.66); T 0.78 (0.61–1.04) *; SNAC 1.07 (1.01–1.38); TNAC 1.06 (1.01–1.15) arbitrary units; * p < 0.05 vs S]. NADPH oxidase subunits did not differ between TNAC, SNAC, and S groups.

Conclusion

N-acetylcysteine supplementation alone reduces oxidative stress in untreated spontaneously hypertensive rats. The combination of N-acetylcysteine and concurrent exercise further decreases oxidative stress. However, the lower oxidative stress does not translate into improved cardiac remodeling and function in untreated spontaneously hypertensive rats.

Introduction

Systemic arterial hypertension is one of a major preventable cause of premature death in developed countries [1]. It is associated with functional and structural changes in target organs with an increased risk of cardiovascular events [1]. Pressure overload induced by arterial hypertension leads to cardiac remodeling, characterized by changes in heart size, shape, and function, that manifest clinically as left ventricular (LV) diastolic and systolic dysfunction [2]. Increased oxidative stress plays an important role in the pathophysiology of arterial hypertension and cardiac remodeling, contributing to myocardial contractile dysfunction [3–7].

Both European and American guidelines on exercise practice in patients with cardiovascular disease recommend regular physical exercise as a first-line non-pharmacological therapy [8, 9]. Regular exercise induces several systemic and cardiac benefits. These include a reduction in peripheral vascular resistance and blood pressure levels, and improvement in physical capacity, body composition, insulin resistance, endothelial function, quality of life and antioxidant properties, with increased antioxidant enzyme activity and stimulation of oxidative stress protection pathways [10–19].

The influence of antioxidant drugs on hypertension-induced cardiac remodeling has received little attention. Glutathione (L-γ glutamyl-cysteinyl-glycine) is an endogenous tripeptide, with an important role in cellular defense against oxidative stress [20, 21]. N-acetylcysteine (NAC) is a precursor of glutathione, which can restore total glutathione levels in the myocardium and reduce oxidative stress markers [22, 23]. Therefore, the combination of NAC and concurrent physical exercise may optimize the cardioprotective response, attenuating cardiac remodeling in hypertensive rats. Despite NAC supplementation being safe and well tolerated, studies are necessary to clarify the relevance of its use under different clinical scenarios such as systemic arterial hypertension and cardiac remodeling [24]. In this study, we evaluated the effects of concurrent training isolated or in combination with NAC supplementation on cardiac remodeling and oxidative stress in spontaneously hypertensive rats (SHR).

Materials and methods

Experimental Groups

Six-month-old male SHR were purchased from the Multidisciplinary Center for Biological Investigation in Laboratory Animal Science, CEMIB, University of Campinas, UNICAMP, SP, Brazil. All experiments and procedures were approved by the Animal Research Ethics Committee, Botucatu Medical School, Sao Paulo State University, UNESP, SP, Brazil (protocol number 1162/2015).

Rats were housed in cages (2 rats/cage) at Botucatu Medical School Experimental Research Unit (UNIPEX). Rats received ad libitum regular chow and water and were maintained under controlled temperature (24 ± 2 °C) and photoperiod (12/12 h light/dark cycles). The animals were divided into four groups: SHR (S, n = 12); SHR treated with N-acetylcysteine (SNAC, n = 13); SHR subjected to concurrent training (T, n = 13); and SHR subjected to concurrent training and N-acetylcysteine (TNAC, n = 14). Rats from SNAC and TNAC groups received 120 mg/kg/day NAC added to the rat chow for 8 weeks [24]. N-acetylcysteine was purchased from Sigma (St. Louis, MO, US). Animals from T and TNAC were subjected to concurrent training three times a week, every other day, for 8 weeks.

Systolic arterial pressure, functional capacity and maximum load carrying capacity were assessed before and after the experimental period. Systolic arterial pressure was measured by plethysmography using the tail-cuff method (Narco Bio-System®, model 709–0610, International Biomedical, Inc, USA) [25].

Maximum load carrying capacity

Resistance training was performed on a ladder designed for rats (110 cm high, 18 cm wide, 2 cm between steps, 80° inclination). All rats were adapted to the environment and encouraged to climb the stairs without load for one week. Upon reaching the top of the ladder, the rats had two minutes to recover and then were stimulated to climb again by manual stimulation. The procedure was repeated until the rats climbed the ladder three times without stimulation. Two days after adaptation, maximum load capacity was established for each rat. The test consisted of stair climbing with progressively increasing loads. Initially, 75% of the animal’s body weight was added and increased by 15% until a load was reached where the rat could not fully climb the ladder [26]. The highest load with which the rat was able to climb the entire length of the ladder was considered the maximum load.

Functional capacity test

One week before the aerobic capacity test, the rats from all groups were adapted to the treadmill for 10 min/day. Functional capacity was evaluated one day after the maximum load test, by physical effort tolerance test. The test consisted of running on a treadmill at an initial speed of 6 m/min, incremented by 3 m/min every three minutes until the animal reached exhaustion. The rats were considered exhausted when they refused to run even after manual or electrical stimulation or were unable to coordinate steps. Maximum running speed and time were recorded, and total distance covered was calculated [15].

Resistance training protocol

Resistance training was performed three times a week, every other day, for 8 weeks, with a gradual load increase [27].

The adaptation period lasted for one week. Rats climbed the stairs three times without stimulation. On the first day, the ascents were without load; on the second and third day, loads of 15% and 30% rat body weight were added, respectively. From the second week to the end of the experimental period, training consisted of four ascents with loads corresponding to 50%, 75%, 90%, and 100% of the maximum load established for each rat, with a two-min recovery period (Table 1).

Table 1.

Physical exercise protocol

Resistive training
Weeks	Days		Load		Number of climbings
1st	1º		0		3
	2º		15% of body weight		3
	3º		30% of body weight		3
2nd to 8th			50, 75, 90 and 100% of maximum load		4
Aerobic training
Weeks			Speed (m/min)		Time (min)
1st			7.5–10		20
2nd			12–14		30
3rd			15–17		40
4th to 8th			~ 19 (60% of Vmax)		40

m Meter, min Minute, Vmax Maximal speed achieved during the functional capacity test

Aerobic training protocol

After resistance training, the rats were subjected to aerobic training on a treadmill [28]. They underwent a gradual increase in training speed and duration over a three-week adaptation period (Table 1). From the fourth week to the end of the experiment, rats were subjected to 40 min running sessions at 60% of the maximum speed reached in the physical effort tolerance test.

Echocardiography

Cardiac structure and LV function were evaluated by echocardiogram and tissue Doppler imaging (TDI) using a commercially available echocardiograph (General Electric Medical Systems, Vivid S6 model, Tirat Carmel, Israel) equipped with a 5–11.5 MHz multifrequency probe as previously described [29–31]. After anesthesia with ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (1 mg/kg) intramuscular, rats were placed in left lateral position. In 2-D parasternal short-axis view, guided M-mode tracings were acquired at or just below the tip of mitral valve leaflets and at the level of the aortic valve and left atrium. The following cardiac structures were measured: left atrium diameter (LA), LV diastolic and systolic diameters (LVDD and LVSD, respectively), LV diastolic posterior wall thickness (DPWT), and aortic diameter (AO). LV relative wall thickness (RWT) was calculated according to the formula: (2 × PWT)/LVDD. LV function was assessed by endocardial fractional shortening (EFS), posterior wall shortening velocity (PWSV), early and late diastolic mitral inflow velocities (E and A waves), E/A ratio, and isovolumetric relaxation time (IVRT). Global LV function was evaluated using the myocardial performance index (Tei index). The study was complemented by TDI evaluation of systolic (S’), early diastolic (E’), and late diastolic (A’) velocity of the mitral annulus (average of lateral and septal walls velocities) and E/TDI E’ ratio [32].

Left ventricle and tissues harvesting

One day after echocardiogram analysis, the rats were euthanized under anesthesia with intraperitoneal thiopental (50 mg/kg). After removing the heart from the chest, atria and ventricles were dissected, weighed separately, frozen in liquid nitrogen and stored at − 80 °C. Lung and liver fragments were collected to determine wet/dry weight ratio, which reflects the amount of liquid in the tissue. Wet/dry weight ratio was also calculated for atria and ventricles.

Antioxidant enzyme activities and lipid hydroperoxide concentration

LV samples (200 mg) were homogenized in 5 mL of cold 0.1 M phosphate buffer, pH 7.0, in a motor-driven Teflon glass Potter–Elvehjem homogenizer. The homogenate was centrifuged at 10,000 g, for 15 min at 4 °C. The supernatant was assayed for total protein, lipid hydroperoxide, and glutathione peroxidase (GSH-Px, EC 1.11.1.9), catalase (EC 1.11.1.6.), and total superoxide dismutase (SOD, EC 1.15.1.1.) activities by spectrophotometry. Enzyme activities were analyzed at 25 °C using a microplate reader (Quant-MQX 200) with KC junior software for computer system control (Bio-Tech Instruments, Winooski, Vermont, USA). Spectrophotometric analyzes were performed in a Pharmacia Biotech spectrophotometer with controlled temperature cuvette chamber (UV/visible Ultrospec 5000 with Swift II applications software for computer system control, Cambridge, UK). All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) [33].

Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Gene expression of NADPH oxidase subunits (Nox2, Nox4, p22 phox, and p47 phox) was analyzed by RT-PCR according to a previously described method [34–37]. Total RNA was extracted from LV myocardium with TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and treated with DNase I (Invitrogen Life Technologies). One microgram of RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit, according to standard methods (Applied Biosystems, Foster City, CA, USA). Aliquots of cDNA were then subjected to real-time PCR using a customized assay containing sense and antisense primers and Taqman (Applied Biosystems, Foster City, CA, USA) probes specific to each gene: Nox2 (Rn00576710_m1), Nox4 (Rn00585380_m1), p22 phox (Rn00577357_m1), and p47 phox (Rn00586945_m1). Amplification and analysis were performed using a Step One Plus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Expression data were normalized to the reference genes: cyclophilin (Rn00690933_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rn01775763_g1). Reactions were performed in triplicate and expression levels calculated using the comparative CT method (2^−ΔΔCT).

Statistical analysis

Data are expressed as mean ± standard deviation or median and percentiles. The Shapiro–Wilk test was performed to test data normality. Comparisons between groups were performed by two-way ANOVA and Bonferroni test, or Kruskal–Wallis and Dunn test. Statistical analyses were performed using Sigma Plot 12.0 (Microsoft). Significance level was set at 5%.

Results

No rat died during the experiment. All rats were subjected to functional evaluation (functional and maximum load carrying capacity, and echocardiogram) and 8–10 animals were randomly chosen for biochemical and molecular analyzes. Systolic blood pressure did not differ between groups in both periods, before and after training (Fig. 1). At the end of the experiment, systolic blood pressure was lower in SNAC and TNAC groups when comparing to the initial period.

Fig. 1.

Systolic blood pressure before (Initial) and after training (Final) period. Data are mean ± SD. S: spontaneously hypertensive rats (SHR, n = 12); T: SHR subjected to concurrent training (n = 13); SNAC: SHR treated with N-acetylcysteine (n = 13); TNAC: SHR subjected to concurrent training and N-acetylcysteine (n = 14). ANOVA and Bonferroni; † p < 0.05 initial period

Figure 2 shows that the exercise protocol was effective in improving functional and maximum load carrying capacity in both trained groups.

Fig. 2.

Functional capacity test and maximum load carrying capacity. A and B initial and final running distance, respectively; C and D initial and final running time, respectively; E and F initial and final maximum carrying load capacity, respectively. Data are mean ± SD and individual values. S: spontaneously hypertensive rats (SHR, n = 12); T: SHR subjected to concurrent training (n = 13); SNAC: SHR treated with N-acetylcysteine (n = 13); TNAC: SHR subjected to concurrent training and N-acetylcysteine (n = 14). ANOVA and Bonferroni; * p < 0.05 vs S; # p < 0.05 vs T; § p < 0.05 vs SNAC

Echocardiogram showed that LV diastolic posterior wall thickness (DPWT) was higher in TNAC than T. Relative wall thickness (RWT) was higher in TNAC than T and SNAC (Table 2). Diastolic function was better in T than S, characterized by a lower E/TDI E’ ratio in the T group. Systolic function was better in TNAC than T, demonstrated by a higher PWSV in the TNAC group (Table 3). Functional parameters did not differ between TNAC and SNAC groups.

Table 2.

Echocardiographic structural data

	S (n = 12)	T (n = 13)	SNAC (n = 13)	TNAC (n = 14)
BW (g)	369 ± 14.7	366 ± 23.7	371 ± 23.3	365 ± 26.8
LVDD (mm)	7.43 ± 0.34	7.72 ± 0.39	7.85 ± 0.46*	7.54 ± 0.49
LVDD/BW (mm/kg)	20.2 ± 1.07	21.2 ± 1.37	21.3 ± 1.64	20.7 ± 1.17
LVSD (mm)	3.49 ± 0.47	3.71 ± 0.32	3.92 ± 0.54*	3.60 ± 0.46
DPWT (mm)	1.50 ± 0.12	1.52 ± 0.10	1.56 ± 0.12	1.62 ± 0.14#
RWT	0.40 ± 0.04	0.39 ± 0.03	0.40 ± 0.04	0.43 ± 0.04#§
AO (mm)	4.38 (4.20–4.38)	4.38 (4.26–4.56)	4.38 (4.29–4.47)	4.29 (4.20–4.38)
LA (mm)	5.92 ± 0.44	5.83 ± 0.38	5.86 ± 0.35	5.95 ± 0.41
LA/AO	1.37 ± 0.11	1.33 ± 0.10	1.34 ± 0.08	1.37 ± 0.07
LA/BW (mm/kg)	16.1 ± 1.04	16.0 ± 1.06	15.9 ± 1.02	16.3 ± 0.82

Data are mean ± SD or median and percentiles

S Spontaneously hypertensive rats (SHR), T SHR subjected to concurrent training, SNAC SHR treated with N-acetylcysteine, TNAC SHR subjected to concurrent training and N-acetylcysteine, EFS Endocardial fractional shortening, PWSV Posterior wall shortening velocity, Tei index Myocardial performance index, TDI S’ Tissue Doppler imaging (TDI) of mitral annulus systolic velocity, E/A Ratio between early (E)-to-late (A) diastolic mitral inflow, IVRT Isovolumetric relaxation time, TDI E’ and TDI A’ TDI of early (E’) and late (A’) diastolic velocity of mitral annulus

Two-way ANOVA and Bonferroni or Kruskal–Wallis and Dunn; *p < 0.05 vs S; # p < 0.05 vs T; § p < 0.05 vs SNAC

Table 3.

Echocardiographic data of left ventricular function

	S (n = 12)	T (n = 13)	SNAC (n = 13)	TNAC (n = 14)
HR (beats/minute)	232 (215–261)	235 (211–267)	244 (222–258)	253 (229–287)
EFS (%)	52.7 ± 5.14	51.9 ± 3.94	50.5 ± 5.41	52.4 ± 4.91
PWSV (mm/s)	39.4 ± 5.01	36.4 ± 2.96	39.7 ± 3.44	41.6 ± 3.57#
Tei index	0.60 ± 0.10	0.56 ± 0.06	0.57 ± 0.09	0.58 ± 0.07
TDI S’ (average, cm/s)	3.50 ± 0.30	3.45 ± 0.30	3.47 ± 0.30	3.57 ± 0.27
Mitral E (cm/s)	91.5 ± 8.05	86.8 ± 7.37	93.5 ± 7.87	89.9 ± 6.62
Mitral A (cm/s)	36.0 (35.0–48.0)	41.0 (36.5–52.0)	47.0 (44.0–53.5)	46.0 (39.0–58.0)
E/A	2.32 ± 0.47	2.13 ± 0.57	2.02 ± 0.46	1.94 ± 0.52
IVRT (ms)	31.3 ± 3.52	33.0 ± 3.37	31.7 ± 2.84	31.9 ± 3.39
TDI E’ (average, cm/s)	3.66 ± 0.38	3.95 ± 0.57	4.10 ± 0.46*	3.93 ± 0.31
TDI A’ (average, cm/s)	2.58 ± 0.51	3.31 ± 0.98*	3.01 ± 0.56	3.20 ± 0.60
Mitral E/TDI E’ average	25.1 ± 2.18	22.4 ± 3.94*	23.0 ± 2.68	23.2 ± 1.98

Data are mean ± SD or median and percentiles

S Spontaneously hypertensive rats (SHR), T SHR subjected to concurrent training, SNAC SHR treated with N-acetylcysteine, TNAC SHR subjected to concurrent training and N-acetylcysteine, HR Heart rate, EFS Endocardial fractional shortening, PWSV Posterior wall shortening velocity, Tei index Myocardial performance index, TDI S’ Tissue Doppler imaging (TDI) of mitral annulus systolic velocity, E/A Ratio between early (E)-to-late (A) diastolic mitral inflow, IVRT Isovolumetric relaxation time, TDI E’ and TDI A’ TDI of early (E’) and late (A’) diastolic velocity of mitral annulus

Two-way ANOVA and Bonferroni or Kruskal–Wallis and Dunn; * p < 0.05 vs S; # p < 0.05 vs T

Anatomical parameters are shown in Table 4. The weight of right ventricle and atria, in absolute and indexed to body weight values, was higher in T and SNAC than S, and did not differ from TNAC. RV wet weight/dry weight ratio was higher in T than S. Atria wet/dry weight ratio was higher in T and SNAC than S. LV weight-to-body weight ratio was higher in TNAC than SNAC.

Table 4.

Anatomical data

	S (n = 12)	T (n = 13)	SNAC (n = 13)	TNAC (n = 14)
BW (g)	369 ± 15	366 ± 24	370 ± 24	365 ± 26
LVW (g)	1.06 ± 0.08	1.09 ± 0.06	1.01 ± 0.13	1.07 ± 0.08
LVW/BW (g/kg)	2.87 ± 0.18	3.00 ± 0.13	2.72 ± 0.32	2.94 ± 0.33§
RVW (g)	0.21 ± 0.03	0.24 ± 0.02*	0.24 ± 0.04*	0.24 ± 0.03
RVW/BW (g/kg)	0.57 ± 0.07	0.65 ± 0.05*	0.65 ± 0.11*	0.66 ± 0.11
Atria weight (g)	0.07 (0.07–0.09)	0.11 (0.10–0.12)*	0.10 (0.08–0.12)*	0.10 (0.09–0.11)
Atria weight/BW (g/kg)	0.20 (0.18–0.23)	0.31 (0.28–0.32)*	0.28 (0.21–0.31)*	0.27 (0.23–0.29)
LV wet/dry weight	3.71 (2.82–3.81)	3.88 (3.54–4.09)	4.27 (3.49–4.69)	4.00 (3.68–4.21)
RV wet/dry weight	3.99 (3.56–4.27)	4.42 (4.25–4.70)*	4.12 (3.89–4.25)	4.16 (4.11–4.32)
Atria wet/dry weight	3.73 ± 0.66	4.34 ± 0.43*	4.32 ± 0.70*	4.75 ± 0.58
Lung wet/dry weight	4.40 (4.18–4.47)	4.49 (4.40–4.56)	4.45 (4.26–4.57)	4.54 (4.38–4.63)
Liver wet/dry weight	3.13 (3.07–3.22)	3.21 (3.14–3.43)	3.12 (3.02–3.17)	3.17 (3.12–3.23)

Data are mean ± SD or median and percentiles

S Spontaneously hypertensive rats (SHR), T SHR subjected to concurrent training, SNAC SHR treated with N-acetylcysteine, TNAC SHR subjected to concurrent training and N-acetylcysteine, BW Body weight, LVW Left ventricular weight, RVW Right ventricular weight

Two-way ANOVA and Bonferroni or Kruskal–Wallis and Dunn; * p < 0.05 vs S; § p < 0.05 vs SNAC

Catalase activity was higher in T than S and TNAC groups. Superoxide dismutase and glutathione peroxidase activities did not differ between groups (Fig. 3). Lipid hydroperoxide concentration was lower in SNAC than S, and lower in TNAC than T and SNAC (Fig. 4).

Fig. 3.

Myocardial antioxidant enzymes activity. S: spontaneously hypertensive rats (SHR); T: SHR subjected to concurrent training; SNAC: SHR treated with N-acetylcysteine; TNAC: SHR subjected to concurrent training and N-acetylcysteine; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase. Two-way ANOVA and Bonferroni; * p < 0.05 vs S; # p < 0.05 vs T

Fig. 4.

Myocardial lipid hydroperoxide (LOOH) concentration. S: spontaneously hypertensive rats (SHR); T: SHR subjected to concurrent training; SNAC: SHR treated with N-acetylcysteine; TNAC: SHR subjected to concurrent training and N-acetylcysteine. Two-way ANOVA and Bonferroni; * p < 0.05 vs S; # p < 0.05 vs T; § p < 0.05 vs SNAC

Gene expression of NADPH oxidase subunits is shown in Fig. 5 and Table 5. Gene expression of the transmembrane p22 phox and Nox2 isoform was higher, and p47 phox lower in T compared to S. Nox4 did not differ between groups.

Fig. 5.

Gene expression of the NADPH subunits. Data are mean ± SD or median and percentiles. S: spontaneously hypertensive rats (SHR, n = 10); T: SHR subjected to concurrent training (n = 10); SNAC: SHR treated with N-acetylcysteine (n = 10); TNAC: SHR subjected to concurrent training and N-acetylcysteine (n = 9); a.u.: arbitrary units. Two-way ANOVA and Bonferroni or Kruskal–Wallis and Dunn; * p < 0.05 vs S

Table 5.

Gene expression of the NADPH subunits

	S (n = 10)	T (n = 10)	SNAC (n = 10)	TNAC (n = 9)
p22 phox (a.u.)	0.93 ± 0.46	1.31 ± 0.52*	1.17 ± 0.31	1.24 ± 0.22
p47 phox (a.u.)	1.37 (0.66–1.66)	0.78 (0.61–1.04)*	1.07 (1.01–1.38)	1.06 (1.01–1.15)
Nox2 (a.u.)	0.81 (0.55–1.25)	2.00 (1.23–3.16)*	1.31 (0.65–1.75)	1.76 (1.29–2.00)
Nox4 (a.u.)	0.74 (0.65–1.00)	0.51 (0.30–0.91)	0.75 (0.51–0.88)	0.68 (0.60–0.87)

Data are mean ± SD or median and percentiles

S Spontaneously hypertensive rats (SHR), T SHR subjected to concurrent training, SNAC SHR treated with N-acetylcysteine, TNAC SHR subjected to concurrent training and N-acetylcysteine, a.u. Arbitrary units

Two-way ANOVA and Bonferroni or Kruskal–Wallis and Dunn; * p < 0.05 vs S

Discussion

In this study, we evaluated the effects of concurrent training alone or combined with NAC administration on cardiac structure and function in spontaneously hypertensive rats.

NAC doses from 120 to 250 mg/kg/day added to rat chow have been used in literature and our laboratory [38–41]. Adamy et al. [38] showed that 120 mg/kg/day oral NAC administered to post-myocardial infarction rats improves cardiac tissue and function. On the other hand, contrary to the common belief that NAC functions solely as an antioxidant drug, excess glutathione from NAC treatment may result in reductive stress with pro-oxidative consequences in mitochondria and increased levels of ROS [42]. As this effect was observed in cell culture, dose associated with NAC-induced reductive stress is not known. Therefore, we opted to use a relatively low NAC dose that was associated with beneficial effects on cardiac remodeling in rats [38].

Current evidence shows that physical exercise improves functional capacity and promotes hemodynamic benefits in experimental animals and hypertensive individuals [43]. Although concurrent training has been strongly recommended in guidelines on exercise prescription [8, 9], its effects in cardiovascular disease rodents have not been established. In this study, we applied a moderate-intensity aerobic exercise protocol due to the untreated hypertensive status. The protocol was effective in increasing functional capacity and maximum load carrying capacity in both trained groups.

Systolic blood pressure did not differ between groups before and after training. However, when comparing initial and final periods, we observed that both SNAC and TNAC had a slight reduction in blood pressure. The fact that arterial hypertension is associated with increased oxidative stress [4] may justify the lower blood pressure in the NAC treated rats. Experimental studies have also shown slight reduction in blood pressure after NAC administration in rats subjected to chronic pressure overload [44, 45]. The effect of physical exercise on SHR blood pressure is controversial; some researchers have found an anti-hypertensive effect of exercise [46], while others observed unaltered [47] or even impaired [48] hypertension.

Echocardiogram showed that SNAC had larger LV diameters than S. We previously observed that NAC administration for 8 weeks did not modulate cardiac remodeling in aortic stenosis rats [41]. Although additional studies are needed to clarify NAC effects on pressure overloaded rat hearts, our data suggest that NAC administration induced LV eccentric remodeling. On the other hand, concurrent training combined with NAC administration induced additional LV concentric hypertrophy, characterized by a higher relative LV wall thickness compared to isolated NAC administration (SNAC) or training (T), and higher LV diastolic posterior wall thickness compared to T.

Adult spontaneously hypertensive rats (SHR) present genetic arterial hypertension similar to human hypertension. At one month old, SHR start to develop hypertension and concentric LV hypertrophy [25]. Despite the persistent elevated pressure, concentric hypertrophy is associated with preserved cardiac performance for long period [25]. Cardiac decompensation usually begins at 18–24 months of age [49, 50]. Therefore, in this study, we may assume that our SHR had LV concentric hypertrophy and preserved ventricular function. Thus, our data suggest that NAC alone induced LV dilation while combined NAC and exercise induced additional concentric hypertrophy.

The posterior wall shortening velocity, a commonly used index of systolic function in rodents, was higher in TNAC than T. Improved systolic function may be observed during concentric remodeling and hypertrophy in rodents [51]. Functional parameters did not differ between the TNAC and SNAC groups. Aerobic exercise can have a dual effect on cardiac remodeling during sustained hypertension. Intensive running in a wheel for long periods had deleterious effects by impairing LV relaxation [52], dilating LV and reducing systolic function [53] in untreated SHR. On the other hand, low intensity aerobic exercise attenuated cardiac remodeling and improved functional capacity and diastolic function in adult SHR [15]. In this study, except for the improved index of diastolic function E/TDI E’, cardiac remodeling did not differ between T and S groups. These data suggest that concurrent training is safe in SHR, although not associated with an improvement in cardiac remodeling.

Oxidative stress is characterized by an imbalance between reactive oxygen species (ROS) production and antioxidant defenses. Lipid hydroperoxide (LOOH) concentration was assessed as a marker of oxidative stress. LOOH concentration was lower in both SNAC and TNAC. NAC supplementation exerts a protective effect against oxidative stress in various diseases, including cardiac failure [20, 40, 41]. NAC is a source of cysteine to glutathione synthesis, which plays an essential role in cell defense against oxidative stress [20]. During cardiac remodeling and heart failure, myocardial total glutathione concentration is reduced [40, 41]. Regular physical exercise increases skeletal muscle ROS production at low levels, which causes a tolerable injury degree, which in turn, up-regulates endogenous antioxidants and stimulates oxidative damage repair systems [13]. Therefore, there is a concern that antioxidant supplementation may prevent beneficial effects of exercise on systemic oxidative status. It was therefore interesting to observe that the combined NAC administration and training further decreased LOOH concentration. NAC supplementation also reduced serum, myocardial, and skeletal muscle oxidative stress in infarcted and aortic stenosis rats [40, 41, 54].

We next assessed the antioxidant activity of superoxide dismutase, glutathione peroxidase, and catalase. In T group, a non-significant reduction in LOOH concentration was combined with increased catalase activity. Both SNAC and TNAC groups had unchanged antioxidant enzyme activity, despite reduced LOOH levels. Thus, our data show that training alone was beneficial for improving myocardial antioxidant capacity; however, the increased catalase activity did not reduce LOOH concentration. Similar results have been seen in exercised SHR [15]. Despite reducing oxidative stress, NAC decreased catalase activity in the TNAC relative to the training group. Therefore, additional studies are needed to clarify the role of combined antioxidant supplementation and exercise on oxidative stress. Although superoxide dismutase activity is usually modulated by physical exercise, contradictory results have been observed in hypertensive rodents subjected to exercise [55, 56]. Similarly to our results, exercise did not change superoxide dismutase activity in rats with arterial hypertension induced by L-NAME [56].

ROS production by NADPH oxidase subunits is involved in the pathophysiology of arterial hypertension and cardiac remodeling [57, 58]. To assess the potential role of NADPH oxidase in increasing oxidative stress, gene expression was analyzed for the Nox2 and Nox4 subunits and the main cardiac isoforms p22 phox (transmembrane protein) and p47 phox (cytosolic regulatory subunit). After binding to the regulatory p22 phox and p47 phox units, Nox2 drives adverse myocardial signaling, while the constitutively active Nox4 is associated with protecting signaling [59]. It was therefore unexpected to observe that Group T had increased Nox2 and p22 phox and decreased p47 phox expression. These data may explain the reason that the increased catalase activity in Group T did not reduce LOOH concentration. The effects of exercise on the myocardial expression of NAPDH subunits under different cardiac injury situations are not clear. A similar pattern of NADPH changes was observed in the myocardium of infarcted rats subjected to isolated resistance exercise for 12 weeks [33]. On the other hand, aerobic exercise reduced Nox2, Nox4, and p22 phox gene expression in the diaphragm of aortic stenosis rats [60]. No differences between TNAC, SNAC and S were observed.

The following data from our study have a potential to be translated into clinical practice: 1) although not associated with improved cardiac remodeling, concurrent training is safe during uncontrolled hypertension; 2) NAC administration reduces oxidative stress with no improvement in cardiac remodeling; 3) combined NAC administration and concurrent training further reduces oxidative stress but has the potential to additionally induce concentric LV hypertrophy in untreated hypertension. A limitation of this study is that citrate synthase activity was not evaluated in the rat skeletal muscle. This assay could have confirmed that exercise training had a similar effect in the exercised groups. Another limitation is the fact that NADPH oxidase subunit activity was not analyzed. Thus, additional research is necessary to clarify whether gene expression of NADPH oxidase subunits express their subunits activity.

Conclusion

N-acetylcysteine supplementation alone reduces oxidative stress in untreated spontaneously hypertensive rats. The combination of N-acetylcysteine and concurrent exercise further decreases oxidative stress. However, the lower oxidative stress does not translate into improved cardiac remodeling and function in untreated spontaneously hypertensive rats.

Authors’ contributions

Conceptualization, A.J. and K.O.; methodology, A.J., K.O. and M.J.G., T.H.D.P., F.C.D., A.R.R.L., E.A.R., G.L.P., L.U.P., I.D., A.A.H.F.; statistical analysis, A.J., K.O. and S.A.O.J.; writing—original draft preparation, A.J., F.L.P., and K.O.; writing—review and editing, A.J., F.L.P., K.O. and M.P.O.; supervision, K.O.; project administration, A.J.; funding acquisition, K.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by CNPq (Proc. n. 307703/2022–3 and 307280/2022–5), FAPESP (Proc. n. 2021/10923–5) and PROPe, UNESP.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All experiments and procedures were approved by the Ethics Committee of Botucatu Medical School, UNESP (Protocol n. 1162/2015). The study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and this study was carried out in compliance with the ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.