Nandrolone decanoate induces cardiac and renal remodeling in female rats, without modification in physiological parameters: The role of ANP system Girlandia Alexandre Brasil, Ewelyne Miranda de Lima, Andrews Marques do Nascimento, Izabela Facco Caliman, Ana Raquel Santos de Medeiros, Mauro S´ergio Batista Silva, Gl´aucia Rodrigues de Abreu, Adelina Martha dos Reis, Tadeu Uggere de Andrade, Nazar´e Souza Bissoli PII: DOI: Reference:
S0024-3205(15)00357-4 doi: 10.1016/j.lfs.2015.07.005 LFS 14441
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
20 February 2015 28 May 2015 3 July 2015
Please cite this article as: Brasil Girlandia Alexandre, de Lima Ewelyne Miranda, do Nascimento Andrews Marques, Caliman Izabela Facco, de Medeiros Ana Raquel Santos, Silva Mauro S´ergio Batista, de Abreu Gl´ aucia Rodrigues, Reis Adelina Martha dos, de Andrade Tadeu Uggere, Bissoli Nazar´e Souza, Nandrolone decanoate induces cardiac and renal remodeling in female rats, without modification in physiological parameters: The role of ANP system, Life Sciences (2015), doi: 10.1016/j.lfs.2015.07.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Nandrolone decanoate induces cardiac and renal remodeling in female rats, without modification in physiological parameters: the role of ANP system
T
Girlandia Alexandre Brasila; Ewelyne Miranda de Limaa; Andrews Marques do
IP
Nascimentoa; Izabela Facco Calimana; Ana Raquel Santos de Medeirosa,b Mauro
SC R
Sérgio Batista Silvac, Gláucia Rodrigues de Abreua; Adelina Martha dos Reisc; Tadeu Uggere de Andraded; Nazaré Souza Bissolia *. a
NU
Department of Physiological Sciences, Federal University of Espirito Santo,Vitória,
ES, Brazil; b
Biological and Health Sciences, Federal Institute of Espirito Santo, Vila Velha, ES,
c
MA
Brazil;
Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo
Horizonte, MG, Brazil. d
D
DepartmentofPharmaceuticalSciences, Universityof Vila Velha, Vila Velha, ES,
TE
Brazil. *
CE P
Address for correspondence: Nazaré Souza Bissoli, PhD. Departamento de Ciências
Fisiológicas, Universidade Federal do Espírito Santo - UFES. Avenida Marechal Campos, 1468 Maruípe, Vitória - ES, 29043-900, Brazil. Tel: +55 (27) 3335-7333,
AC
Fax: +55 (27) 3335-7340, E-mail: [email protected].
ACCEPTED MANUSCRIPT
2
ABSTRACT Anabolic-androgenic steroids are misused, including women, but little is known about the cardiovascular effects of these drugs on females. Aim: Evaluated the effects of
T
nandrolone decanoate (ND), physical exercise and estrogen deficiency on female
IP
rats. Main methods: Female Wistar rats were divided into 8 groups: S and OVX:
SC R
(SHAM: sham surgery; OVX: ovariectomy), SE and OVXE (resistance exercise 5 times a week), SD and OVXD (treated with ND, 20 mg/kg/week for 4 weeks); and SDE and OVXDE. Treatments were initiated 21 days after surgery. The Bezold-
NU
Jarisch reflex was assessed by Phenylbiguanide administration. The right atrium, kidney, and serum were collected for molecular analyses by RT-PCR of atrial natriuretic peptide (ANP), A-type natriuretic peptide receptor (NPR-A) and NPR-C.
MA
ELISA assay to estradiol and testosterone concentrations. The gastrocnemius muscle, heart and kidney weights/tibia length were measured. Morphometric analysis of heart was made (H/E) and collagen content of heart and kidney were evaluated
D
using Pirossirius Red. Key findings: ND treatment increased ANP expression on
TE
atrium and decreased NPR-A expression in kidney. Physical exercise and ovariectomy did not alter this parameter. NPR-C level was reduced in the SDE and
CE P
OVXDE. Renal and cardiac hypertrophy was observed after ND treatment, with collagen deposition. Plasma estrogen concentrations were reduced and serum testosterone concentrations were increased after ND treatment. Significance: ANP
AC
has an important role in modulating the cardiovascular effects of ND in females. This modulating may have occurred by the increasing ANP expression, reducing NPR-A and NPR-C expression levels, and changing sex hormone levels.
Key words: nandrolone decanoate; cardiovascular system; ANP; resistive training; sex hormones.
ACCEPTED MANUSCRIPT
3
INTRODUCTION The abuse of anabolic-androgenic steroids (AAS) has been considered a serious public health problem, especially among adolescents and young adults of both
T
genders (Amsterdam et al. 2010; Thiblin and Petersson 2005; Gruber and Pope
IP
2000). Most often, individuals misuse these steroids to increase muscle mass and
SC R
improve their physical shape without great concern for the adverse effects that can be caused by these substances (Rashid and Osmerod 2007). The excessive use of AAS can have acute and chronic side effects on different tissues as brain, kidneys,
NU
heart and vasculature (Amsterdam et al. 2010). The cardiovascular system is an important target of the adverse effects of AAS (Fineschi et al. 2001; Hausmann et al. 1998; Dickerman et al. 1995).
MA
Nandrolone decanoate (ND) is one of the most popular AAS (Perry et al. 2005) and has been implicated in increase on blood pressure (Franquni et al 2013; Hassan and Kamal 2013; Bissoli et al. 2009; Tseng et al. 1994), changes in the reflex control of
D
blood pressure (Franquni et al. 2013; Andrade et al. 2011; Bissoli et al. 2009;
TE
Andrade et al. 2008), and several cardiac abnormalities such as hypertrophy, collagen deposition in the extracellular matrix (Tanno et al. 2011; Bissoli et al. 2009;
CE P
Andrade et al. 2008), imbalance in inflammatory cytokines (Riezzo et al. 2014; Franquni et al. 2013), and was associated with genetic damage (Pozzi et al. 2013). At the opposite, physical exercise has been implicated with positive effects on
AC
cardiovascular system, such as protective effects against ischemic events (Le Page et al. 2009; Kruk 2007) and increase on antioxidant enzymes (Chaves et al. 2006) and the use of ND could be related to a loss of the beneficial effects of physical exercise (Chaves et al. 2006; Cunha et al. 2005). Most of the studies involving AAS have been performed with men and/or male animals. However, the misuse of these substances has increase among women (Elliot and Goldberg 2000). Therefore, because there is evidence that AAS alters the female reproductive physiology (Belardin et al. 2014), other studies need to be conducted to elucidate the changes induced by AAS in females, particularly the changes associated with the cardiovascular system and its regulation. The ANP system is one of the most important mechanisms involved in the control of the cardiovascular system and is able to regulate a variety of physiological processes that affect the cardiovascular system (McKie et al. 2010; Holtwick et al. 2003). In addition, changes to the ANP system are widely associated with heart diseases such
ACCEPTED MANUSCRIPT
4
as hypertrophy (Klein et al. 1995; Ruskoahoo 1992) and heart failure (Klein et al. 1995). Our group has demonstrated that high doses of ND alter the reflex control of the
T
cardiovascular system through the Bezold-Jarisch reflex (BJR) in a dose- and time-
IP
dependent manner (Franquni et al. 2013; Andrade et al. 2011; Bissoli et al. 2009).
SC R
These changes were associated with cardiac hypertrophy, one of the factors that can alter the BJR (Bissoli et al. 2009). However, the effect of AAS on this reflex has not yet been tested in females. In addition, it has been demonstrated that cardiac
NU
hypertrophy affects the ANP system (Holtwick et al. 2003; Maisel et al. 2002; Knowles et al. 2001; Kishimoto et al. 2001; Calderone et al. 1998; Saito et al. 1987) and that ANP affects the BJR (Bissoli et al. 2000; Imaizumi et al. 1987; Thorén et al.
MA
1986). Thus, we hypothesized that ND administration to female rats can alter the ANP system and the BJR.
Therefore, the main of this study was to evaluate the effects of ND treatment during
D
resistance training on ANP expression and the BJR in female rats and to assess the
TE
role of estrogen in this process.
Animals
CE P
MATERIALS AND METHODS
Adult female Wistar rats weighing 180-200 g were obtained from the breeding unit at
AC
the Federal University of Espirito Santo. The rats were housed with controlled temperature (22ºC), humidity (50%) and light cycles (12-h light/dark), and they were given water and standard rat chow (Purina Labina, SP-Brazil) ad libitum. The procedures were conducted in compliance with the guidelines for the ethical use of animals in scientific research as stated by the Federation of the Brazilian Society of Experimental Biology and were approved by the Ethics Committee for Animal Use at the Federal University of Espirito Santo (031/2012). Eight groups of female rats were studied (n=7 each): Sham (S); Ovariectomized (OVX); OVX treated with ND (OVXD: 20 mg/kg/week; DecaDurabolin®, Organon Inc., São Paulo, SP, Brazil); SHAM treated with ND (SD); OVX animals submitted to resistance training (RT) 5 days a week (OVXE); SHAM exercised (SE), OVX exercised and treated with ND (OVXDE), and SHAM exercised and treated with ND (SDE). Bilateral ovariectomy was performed in female rats under ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia by intraperitoneal injection (i.p). The
ACCEPTED MANUSCRIPT
5
females were subjected to a muscular incision to open the peritoneal cavity for posterior connection of the uterine tubules and removal of the ovaries. Then, the peritoneal cavity was sutured and cleaned. The female sham groups only underwent
T
an incision. Next, the animals were allowed to recover. The treatments started on the
IP
21st day after bilateral ovariectomy or sham surgery and were performed for 4
SC R
weeks. The treatment was made by intramuscular injection, and the vehicle used was peanut oil. The estrous cycles of the animals were evaluated daily. The experimental protocols were performed when the animals of the S and SE group
NU
were in proestrus, and the animals treated with ND remained in diestrus II. Resistance training
MA
The RT protocol was designed as described by Voltarelli et al. (2002) with modifications. One week before the tests, the animals were adapted to water (31 ± 1ºC) for 60 min every day without exercise training.
D
The RT protocol was as follows: first, the animals performed jump series in the water
TE
(31 ± 1ºC; the water depth corresponded to three times the length of the animal) for 6 minutes (30 s of exercise and 30 s of rest) with a load of 40% of body weight. After a
CE P
period of 9 minutes, the animals performed 12 minutes of exercise (60 s followed by 30 s of rest), with a load of 30% of body weight. After the last series, 25-µL blood samples were obtained from a cut at the tip of the tail to evaluate capillary blood
AC
lactate levels (BM-Lactate® strips and Accutrend® Lactate monitor; Roche, São Paulo, Brazil) to confirm that the exercise was performed under anaerobic conditions (indicated by blood lactate values above 7.0 mmoL/L; (Voltarelli et al. 2002). These protocols were performed 5 days a week for 4 weeks (Cunha et al. 2005). Measurement of mean arterial pressure (MAP), heart rate (HR) and BezoldJarisch reflex evaluation Forty-eight hours after the experimental protocol, MAP and heart rate (HR) were determined by direct measurement in each group at the end of treatment (Bissoli et al. 2009). For this procedure, on the day before the measurement, a catheter filled with saline (PE-50) was inserted into the left femoral artery under anesthesia (ketamine 100 mg/kg, xylazine 10 mg/kg; i.p). The free end of the catheter was exteriorized at the cervical dorsal area. For the MAP measurement, the arterial catheter was attached to a 40-cm polyethylene catheter during the 40-min recording
ACCEPTED MANUSCRIPT
6
period. MAP measurements were obtained in quiet, conscious rats that had complete freedom to move throughout the cage. The MAP was recorded using a pressure transducer coupled to a MP-100 System Guide (model MP100-CE; Biopac Systems,
T
Santa Barbara, CA, USA). The HR was calculated instantaneously from the intervals
IP
of pressure pulses. The Bezold-jarisch reflex was evaluated by the maximum
SC R
bradycardiac and hypotensive responses obtained by in bolus application of phenylbiguanide (1.5-24g/kg). The reduction on HR and diastolic blood pressure (DBP) after each dose was recorded and the percentage of decrease of HR and DBP
NU
was used as reflex sensitivity index (Franquni et. 2013).
Collection of tissues and histological analysis
MA
After measuring MAP and HR, the females were euthanized by decapitation, and the kidney and right atria (RA) were removed, washed with saline, dried with filter paper,
D
weighed, frozen in liquid nitrogen and stored at -80°C. For 17β-estradiol and testosterone analysis, the serum was collected and submitted into ELISA protocol
TE
following the manufactures instructions (DRG International Inc., Springfield, NJ, USA). The ventricles and kidneys were fixed in formalin (10% buffer) for 24 hours,
CE P
after that a section of each organ was embedded in paraffin. A 5 m thick slice of the ventricles was stained with hematoxylin/eosin for morphometric analysis or Picrosirius red for collagen deposition (as described by Franquni et al., 2013). The
AC
kidney was stained with Picrosirius red for analysis of collagen deposition in renal cortex as described by Tachaudomdach et al. 2015. The collagen content was evaluated using the Software Image J®. For Picrossirius analysis, 10 high power fields were evaluated per animal.
Biochemical analysis Three days before the end of the protocol, the animals were kept in a metabolic cage. The 24h urinary volume was collected and measured for two days and then frozen at -80ºC. The sodium urinary contents were evaluated by a flame photometer (Bissoli et al. 2000). After the experimental protocol, the serum of the animals were separated by centrifugation (3000rpm, 4ºC, 15 minutes) and kept into -80ºC. The creatinine and urea evaluation were made using commercial kits (Bioclin®, Belo Horizonte, Minas Gerais, Brazil) according to the manufacturer instructions.
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ANP, NPR-A and NPR-C gene expression by Real Time PCR Total mRNA was extracted from atria and kidneys using a protocol already well established. Tissues were processed in sterile eppendorf tubes with 1 mL of TRIzol®
T
reagent, followed by homogenization. Chloroform was added in the homogenate and
IP
centrifugation was carried out for 15 minutes at 12.000 G/4C. The supernatant was
SC R
transferred to a second eppendorf and with 650L of isopropanol, following centrifugation for 35 minutes at 12.000G/4C. The supernatant was discharged and added 1.3 mL of 75% ethanol to the pellet and a centrifugation was performed for 15
NU
minutes at 12.000 G /4C. Lately, the supernatant was discharged and the extracted RNA was resuspended in 20 L with UltraPure™ DNAse, RNAse free water
MA
(Intrivitrogen Life technologiesTM, Carlsbad, CA, USA) and maintained at -80°C for one day. The RNA was treated with DNase as instructed by the manufacturer (Turbo DNA freeTM, Ambion Inc. Carlsbad, CA, USA). In short, each sample was added
D
10% volume of buffer to 1 L DNase I and DNase I buffer. The samples were
TE
incubated for 30 min at 37C. 10% of the volume of inactivation reagent were added, followed by incubation for 2 min at room temperature. Then, the samples were
CE P
centrifuged at 10.000 rpm for 2 min to precipitate the reagent. The mRNA expression levels of ANP and its receptors (NPR-A and NPR-C) were determined by Real Time PCR. ANP gene expression was evaluated in the RA, and
AC
NPR-A and NPR-C expression levels were determined in the kidney. cDNA was synthesized by the reverse transcription of mRNA. A mix solution containing random primers (100 ηg/µL), deoxyribonucleotide triphosphates mix, dNTP(10 mM) and sterile DNAseRNAse free water to a final reaction volume of 13L were added to 1 g of RNA. Followed by an incubation for 5 minutes to 65ºC and 4ºC for 1 minute, the retrotranscription took PCR 5x buffer, dithiothreitol, DTT (0.1M), ribonucleic inhibitor (40 U/µL) and Superscript III RT enzyme (all reagent and enzymes were purchased from LifeTechnologiesTM Carlsbad, CA, USA). The incubation process occurred at 25⁰ C for 5 minutes, 50°C for 50 minutes and 70⁰ C for15 minutes. The resulting cDNA was stored at -20°C. For quantitative real-time PCR, the amplification of all geneswere normalized by s26 gene amplification (Haroon et al. 2015). The reaction was performed in 96-well plates with SYBR® green master mix (LifeTechnologiesTM). Relative gene expression analysis used 2-CT method described by Livak and Schmittgen (2001), which considered the comparison of different treatments with one
ACCEPTED MANUSCRIPT
8
of those groups with the lower values of threshold cycle (CT). Real-time PCR followed the manufacturer’s procedures (Applied Biosystems, Foster City, CA, USA) in an ABI-Prism 7000 System. using the following cycles: [stage 1], a cycle of 52°C/2 min;
T
[stage 2], a cycle of 95 °C/10 min; [stage 3], 60 cycles of 95 °C/0.15 min and 50 °C/1
IP
min.
SC R
cDNAs for ANP, NPR-A and NPR-C using specific primers (ANP: 5’-GGA TTT CAA GAA CCT GCT AGA-3’ and 5’-CTT CAT CGG TCT GCT CGC TCA-3’; NPR-A: 5’ATC ACA GTG AAT CAC CAG GAG TTC-3’ and 5’-AGA TGT AGA TAA CTC TGC
NU
CCT TTC G-3’; NPR-C: 5’-CCT ACA ATT TCG ACG AGA CCA AA-3’ and 5’-ACT CGC TCA CTG CCC TGG ATG TA-3’). Statistical analysis
MA
Data are expressed as the means standard errors of mean (SEM), and the statistical analysis was performed using GraphPad Prism 5 (Graph Pad Software, La
D
Jolla, CA – USA). Data were compared using one-way analysis of variance (ANOVA) followed by Tukeypost hoc test. For hormones data the results were compared using
TE
one-way analysis of variance (ANOVA) followed by Newman-Keuls. Multiple
RESULTS
CE P
Comparison post hoc test Data were considered statistically significant if p
S0024-3205(15)00357-4 doi: 10.1016/j.lfs.2015.07.005 LFS 14441
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
20 February 2015 28 May 2015 3 July 2015
Please cite this article as: Brasil Girlandia Alexandre, de Lima Ewelyne Miranda, do Nascimento Andrews Marques, Caliman Izabela Facco, de Medeiros Ana Raquel Santos, Silva Mauro S´ergio Batista, de Abreu Gl´ aucia Rodrigues, Reis Adelina Martha dos, de Andrade Tadeu Uggere, Bissoli Nazar´e Souza, Nandrolone decanoate induces cardiac and renal remodeling in female rats, without modification in physiological parameters: The role of ANP system, Life Sciences (2015), doi: 10.1016/j.lfs.2015.07.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Nandrolone decanoate induces cardiac and renal remodeling in female rats, without modification in physiological parameters: the role of ANP system
T
Girlandia Alexandre Brasila; Ewelyne Miranda de Limaa; Andrews Marques do
IP
Nascimentoa; Izabela Facco Calimana; Ana Raquel Santos de Medeirosa,b Mauro
SC R
Sérgio Batista Silvac, Gláucia Rodrigues de Abreua; Adelina Martha dos Reisc; Tadeu Uggere de Andraded; Nazaré Souza Bissolia *. a
NU
Department of Physiological Sciences, Federal University of Espirito Santo,Vitória,
ES, Brazil; b
Biological and Health Sciences, Federal Institute of Espirito Santo, Vila Velha, ES,
c
MA
Brazil;
Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo
Horizonte, MG, Brazil. d
D
DepartmentofPharmaceuticalSciences, Universityof Vila Velha, Vila Velha, ES,
TE
Brazil. *
CE P
Address for correspondence: Nazaré Souza Bissoli, PhD. Departamento de Ciências
Fisiológicas, Universidade Federal do Espírito Santo - UFES. Avenida Marechal Campos, 1468 Maruípe, Vitória - ES, 29043-900, Brazil. Tel: +55 (27) 3335-7333,
AC
Fax: +55 (27) 3335-7340, E-mail: [email protected].
ACCEPTED MANUSCRIPT
2
ABSTRACT Anabolic-androgenic steroids are misused, including women, but little is known about the cardiovascular effects of these drugs on females. Aim: Evaluated the effects of
T
nandrolone decanoate (ND), physical exercise and estrogen deficiency on female
IP
rats. Main methods: Female Wistar rats were divided into 8 groups: S and OVX:
SC R
(SHAM: sham surgery; OVX: ovariectomy), SE and OVXE (resistance exercise 5 times a week), SD and OVXD (treated with ND, 20 mg/kg/week for 4 weeks); and SDE and OVXDE. Treatments were initiated 21 days after surgery. The Bezold-
NU
Jarisch reflex was assessed by Phenylbiguanide administration. The right atrium, kidney, and serum were collected for molecular analyses by RT-PCR of atrial natriuretic peptide (ANP), A-type natriuretic peptide receptor (NPR-A) and NPR-C.
MA
ELISA assay to estradiol and testosterone concentrations. The gastrocnemius muscle, heart and kidney weights/tibia length were measured. Morphometric analysis of heart was made (H/E) and collagen content of heart and kidney were evaluated
D
using Pirossirius Red. Key findings: ND treatment increased ANP expression on
TE
atrium and decreased NPR-A expression in kidney. Physical exercise and ovariectomy did not alter this parameter. NPR-C level was reduced in the SDE and
CE P
OVXDE. Renal and cardiac hypertrophy was observed after ND treatment, with collagen deposition. Plasma estrogen concentrations were reduced and serum testosterone concentrations were increased after ND treatment. Significance: ANP
AC
has an important role in modulating the cardiovascular effects of ND in females. This modulating may have occurred by the increasing ANP expression, reducing NPR-A and NPR-C expression levels, and changing sex hormone levels.
Key words: nandrolone decanoate; cardiovascular system; ANP; resistive training; sex hormones.
ACCEPTED MANUSCRIPT
3
INTRODUCTION The abuse of anabolic-androgenic steroids (AAS) has been considered a serious public health problem, especially among adolescents and young adults of both
T
genders (Amsterdam et al. 2010; Thiblin and Petersson 2005; Gruber and Pope
IP
2000). Most often, individuals misuse these steroids to increase muscle mass and
SC R
improve their physical shape without great concern for the adverse effects that can be caused by these substances (Rashid and Osmerod 2007). The excessive use of AAS can have acute and chronic side effects on different tissues as brain, kidneys,
NU
heart and vasculature (Amsterdam et al. 2010). The cardiovascular system is an important target of the adverse effects of AAS (Fineschi et al. 2001; Hausmann et al. 1998; Dickerman et al. 1995).
MA
Nandrolone decanoate (ND) is one of the most popular AAS (Perry et al. 2005) and has been implicated in increase on blood pressure (Franquni et al 2013; Hassan and Kamal 2013; Bissoli et al. 2009; Tseng et al. 1994), changes in the reflex control of
D
blood pressure (Franquni et al. 2013; Andrade et al. 2011; Bissoli et al. 2009;
TE
Andrade et al. 2008), and several cardiac abnormalities such as hypertrophy, collagen deposition in the extracellular matrix (Tanno et al. 2011; Bissoli et al. 2009;
CE P
Andrade et al. 2008), imbalance in inflammatory cytokines (Riezzo et al. 2014; Franquni et al. 2013), and was associated with genetic damage (Pozzi et al. 2013). At the opposite, physical exercise has been implicated with positive effects on
AC
cardiovascular system, such as protective effects against ischemic events (Le Page et al. 2009; Kruk 2007) and increase on antioxidant enzymes (Chaves et al. 2006) and the use of ND could be related to a loss of the beneficial effects of physical exercise (Chaves et al. 2006; Cunha et al. 2005). Most of the studies involving AAS have been performed with men and/or male animals. However, the misuse of these substances has increase among women (Elliot and Goldberg 2000). Therefore, because there is evidence that AAS alters the female reproductive physiology (Belardin et al. 2014), other studies need to be conducted to elucidate the changes induced by AAS in females, particularly the changes associated with the cardiovascular system and its regulation. The ANP system is one of the most important mechanisms involved in the control of the cardiovascular system and is able to regulate a variety of physiological processes that affect the cardiovascular system (McKie et al. 2010; Holtwick et al. 2003). In addition, changes to the ANP system are widely associated with heart diseases such
ACCEPTED MANUSCRIPT
4
as hypertrophy (Klein et al. 1995; Ruskoahoo 1992) and heart failure (Klein et al. 1995). Our group has demonstrated that high doses of ND alter the reflex control of the
T
cardiovascular system through the Bezold-Jarisch reflex (BJR) in a dose- and time-
IP
dependent manner (Franquni et al. 2013; Andrade et al. 2011; Bissoli et al. 2009).
SC R
These changes were associated with cardiac hypertrophy, one of the factors that can alter the BJR (Bissoli et al. 2009). However, the effect of AAS on this reflex has not yet been tested in females. In addition, it has been demonstrated that cardiac
NU
hypertrophy affects the ANP system (Holtwick et al. 2003; Maisel et al. 2002; Knowles et al. 2001; Kishimoto et al. 2001; Calderone et al. 1998; Saito et al. 1987) and that ANP affects the BJR (Bissoli et al. 2000; Imaizumi et al. 1987; Thorén et al.
MA
1986). Thus, we hypothesized that ND administration to female rats can alter the ANP system and the BJR.
Therefore, the main of this study was to evaluate the effects of ND treatment during
D
resistance training on ANP expression and the BJR in female rats and to assess the
TE
role of estrogen in this process.
Animals
CE P
MATERIALS AND METHODS
Adult female Wistar rats weighing 180-200 g were obtained from the breeding unit at
AC
the Federal University of Espirito Santo. The rats were housed with controlled temperature (22ºC), humidity (50%) and light cycles (12-h light/dark), and they were given water and standard rat chow (Purina Labina, SP-Brazil) ad libitum. The procedures were conducted in compliance with the guidelines for the ethical use of animals in scientific research as stated by the Federation of the Brazilian Society of Experimental Biology and were approved by the Ethics Committee for Animal Use at the Federal University of Espirito Santo (031/2012). Eight groups of female rats were studied (n=7 each): Sham (S); Ovariectomized (OVX); OVX treated with ND (OVXD: 20 mg/kg/week; DecaDurabolin®, Organon Inc., São Paulo, SP, Brazil); SHAM treated with ND (SD); OVX animals submitted to resistance training (RT) 5 days a week (OVXE); SHAM exercised (SE), OVX exercised and treated with ND (OVXDE), and SHAM exercised and treated with ND (SDE). Bilateral ovariectomy was performed in female rats under ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia by intraperitoneal injection (i.p). The
ACCEPTED MANUSCRIPT
5
females were subjected to a muscular incision to open the peritoneal cavity for posterior connection of the uterine tubules and removal of the ovaries. Then, the peritoneal cavity was sutured and cleaned. The female sham groups only underwent
T
an incision. Next, the animals were allowed to recover. The treatments started on the
IP
21st day after bilateral ovariectomy or sham surgery and were performed for 4
SC R
weeks. The treatment was made by intramuscular injection, and the vehicle used was peanut oil. The estrous cycles of the animals were evaluated daily. The experimental protocols were performed when the animals of the S and SE group
NU
were in proestrus, and the animals treated with ND remained in diestrus II. Resistance training
MA
The RT protocol was designed as described by Voltarelli et al. (2002) with modifications. One week before the tests, the animals were adapted to water (31 ± 1ºC) for 60 min every day without exercise training.
D
The RT protocol was as follows: first, the animals performed jump series in the water
TE
(31 ± 1ºC; the water depth corresponded to three times the length of the animal) for 6 minutes (30 s of exercise and 30 s of rest) with a load of 40% of body weight. After a
CE P
period of 9 minutes, the animals performed 12 minutes of exercise (60 s followed by 30 s of rest), with a load of 30% of body weight. After the last series, 25-µL blood samples were obtained from a cut at the tip of the tail to evaluate capillary blood
AC
lactate levels (BM-Lactate® strips and Accutrend® Lactate monitor; Roche, São Paulo, Brazil) to confirm that the exercise was performed under anaerobic conditions (indicated by blood lactate values above 7.0 mmoL/L; (Voltarelli et al. 2002). These protocols were performed 5 days a week for 4 weeks (Cunha et al. 2005). Measurement of mean arterial pressure (MAP), heart rate (HR) and BezoldJarisch reflex evaluation Forty-eight hours after the experimental protocol, MAP and heart rate (HR) were determined by direct measurement in each group at the end of treatment (Bissoli et al. 2009). For this procedure, on the day before the measurement, a catheter filled with saline (PE-50) was inserted into the left femoral artery under anesthesia (ketamine 100 mg/kg, xylazine 10 mg/kg; i.p). The free end of the catheter was exteriorized at the cervical dorsal area. For the MAP measurement, the arterial catheter was attached to a 40-cm polyethylene catheter during the 40-min recording
ACCEPTED MANUSCRIPT
6
period. MAP measurements were obtained in quiet, conscious rats that had complete freedom to move throughout the cage. The MAP was recorded using a pressure transducer coupled to a MP-100 System Guide (model MP100-CE; Biopac Systems,
T
Santa Barbara, CA, USA). The HR was calculated instantaneously from the intervals
IP
of pressure pulses. The Bezold-jarisch reflex was evaluated by the maximum
SC R
bradycardiac and hypotensive responses obtained by in bolus application of phenylbiguanide (1.5-24g/kg). The reduction on HR and diastolic blood pressure (DBP) after each dose was recorded and the percentage of decrease of HR and DBP
NU
was used as reflex sensitivity index (Franquni et. 2013).
Collection of tissues and histological analysis
MA
After measuring MAP and HR, the females were euthanized by decapitation, and the kidney and right atria (RA) were removed, washed with saline, dried with filter paper,
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weighed, frozen in liquid nitrogen and stored at -80°C. For 17β-estradiol and testosterone analysis, the serum was collected and submitted into ELISA protocol
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following the manufactures instructions (DRG International Inc., Springfield, NJ, USA). The ventricles and kidneys were fixed in formalin (10% buffer) for 24 hours,
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after that a section of each organ was embedded in paraffin. A 5 m thick slice of the ventricles was stained with hematoxylin/eosin for morphometric analysis or Picrosirius red for collagen deposition (as described by Franquni et al., 2013). The
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kidney was stained with Picrosirius red for analysis of collagen deposition in renal cortex as described by Tachaudomdach et al. 2015. The collagen content was evaluated using the Software Image J®. For Picrossirius analysis, 10 high power fields were evaluated per animal.
Biochemical analysis Three days before the end of the protocol, the animals were kept in a metabolic cage. The 24h urinary volume was collected and measured for two days and then frozen at -80ºC. The sodium urinary contents were evaluated by a flame photometer (Bissoli et al. 2000). After the experimental protocol, the serum of the animals were separated by centrifugation (3000rpm, 4ºC, 15 minutes) and kept into -80ºC. The creatinine and urea evaluation were made using commercial kits (Bioclin®, Belo Horizonte, Minas Gerais, Brazil) according to the manufacturer instructions.
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ANP, NPR-A and NPR-C gene expression by Real Time PCR Total mRNA was extracted from atria and kidneys using a protocol already well established. Tissues were processed in sterile eppendorf tubes with 1 mL of TRIzol®
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reagent, followed by homogenization. Chloroform was added in the homogenate and
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centrifugation was carried out for 15 minutes at 12.000 G/4C. The supernatant was
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transferred to a second eppendorf and with 650L of isopropanol, following centrifugation for 35 minutes at 12.000G/4C. The supernatant was discharged and added 1.3 mL of 75% ethanol to the pellet and a centrifugation was performed for 15
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minutes at 12.000 G /4C. Lately, the supernatant was discharged and the extracted RNA was resuspended in 20 L with UltraPure™ DNAse, RNAse free water
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(Intrivitrogen Life technologiesTM, Carlsbad, CA, USA) and maintained at -80°C for one day. The RNA was treated with DNase as instructed by the manufacturer (Turbo DNA freeTM, Ambion Inc. Carlsbad, CA, USA). In short, each sample was added
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10% volume of buffer to 1 L DNase I and DNase I buffer. The samples were
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incubated for 30 min at 37C. 10% of the volume of inactivation reagent were added, followed by incubation for 2 min at room temperature. Then, the samples were
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centrifuged at 10.000 rpm for 2 min to precipitate the reagent. The mRNA expression levels of ANP and its receptors (NPR-A and NPR-C) were determined by Real Time PCR. ANP gene expression was evaluated in the RA, and
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NPR-A and NPR-C expression levels were determined in the kidney. cDNA was synthesized by the reverse transcription of mRNA. A mix solution containing random primers (100 ηg/µL), deoxyribonucleotide triphosphates mix, dNTP(10 mM) and sterile DNAseRNAse free water to a final reaction volume of 13L were added to 1 g of RNA. Followed by an incubation for 5 minutes to 65ºC and 4ºC for 1 minute, the retrotranscription took PCR 5x buffer, dithiothreitol, DTT (0.1M), ribonucleic inhibitor (40 U/µL) and Superscript III RT enzyme (all reagent and enzymes were purchased from LifeTechnologiesTM Carlsbad, CA, USA). The incubation process occurred at 25⁰ C for 5 minutes, 50°C for 50 minutes and 70⁰ C for15 minutes. The resulting cDNA was stored at -20°C. For quantitative real-time PCR, the amplification of all geneswere normalized by s26 gene amplification (Haroon et al. 2015). The reaction was performed in 96-well plates with SYBR® green master mix (LifeTechnologiesTM). Relative gene expression analysis used 2-CT method described by Livak and Schmittgen (2001), which considered the comparison of different treatments with one
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of those groups with the lower values of threshold cycle (CT). Real-time PCR followed the manufacturer’s procedures (Applied Biosystems, Foster City, CA, USA) in an ABI-Prism 7000 System. using the following cycles: [stage 1], a cycle of 52°C/2 min;
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[stage 2], a cycle of 95 °C/10 min; [stage 3], 60 cycles of 95 °C/0.15 min and 50 °C/1
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min.
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cDNAs for ANP, NPR-A and NPR-C using specific primers (ANP: 5’-GGA TTT CAA GAA CCT GCT AGA-3’ and 5’-CTT CAT CGG TCT GCT CGC TCA-3’; NPR-A: 5’ATC ACA GTG AAT CAC CAG GAG TTC-3’ and 5’-AGA TGT AGA TAA CTC TGC
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CCT TTC G-3’; NPR-C: 5’-CCT ACA ATT TCG ACG AGA CCA AA-3’ and 5’-ACT CGC TCA CTG CCC TGG ATG TA-3’). Statistical analysis
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Data are expressed as the means standard errors of mean (SEM), and the statistical analysis was performed using GraphPad Prism 5 (Graph Pad Software, La
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Jolla, CA – USA). Data were compared using one-way analysis of variance (ANOVA) followed by Tukeypost hoc test. For hormones data the results were compared using
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one-way analysis of variance (ANOVA) followed by Newman-Keuls. Multiple
RESULTS
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Comparison post hoc test Data were considered statistically significant if p
