Summary
Background
The renin-angiotensin-aldosterone system is known to play an important role in the pathophysiology and development of heart failure. Several studies have reported the benefits of testosterone in heart failure. However, the mechanisms of testosterone-induced effects on heart failure require further study.
Aims
To determine the effects of castration and testosterone administration on cardiac function and angiotensin II receptor function in rats with isoproterenol-induced heart failure.
Methods
Wistar rats were divided randomly into control and heart failure groups. The heart failure groups were further divided into the following groups: castration; castration + testosterone replacement; and sham castration. Echocardiography and haemodynamic measurements were used to evaluate cardiac function. Cardiocyte apoptosis and fibrosis were determined using terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling (TUNEL) staining and Masson’s Trichrome staining, respectively. Angiotensin II receptor (AT1 and AT2) messenger ribonucleic acid (mRNA) expression levels were assayed using real-time reverse transcriptase-polymerase chain reactions, while Western immunoblotting was used to estimate Bcl-2 protein expression levels.
Results
Castration significantly increased cardiomyocyte apoptosis and fibrosis that was normally induced by isoproterenol ( P < 0.05). AT2 receptor mRNA expression in the castration group was increased and Bcl-2 protein expression was decreased compared with the castration + testosterone replacement group ( P < 0.05).
Conclusion
These data suggest that androgen therapy could play an important role in pathophysiological changes in heart failure and have beneficial effects for its treatment.
Résumé
Justification
Le système rénine-angiotensine-aldostérone joue un rôle important dans la physiopathologie et l’apparition de l’insuffisance cardiaque. De nombreuses études ont rapporté les bénéfices de l’administration de testostérone dans l’insuffisance cardiaque. Cependant, les mécanismes des essais induits par la testostérone dans l’insuffisance cardiaque sont mal connus.
Objectif
Déterminer les effets de la castration et de l’administration de testostérone sur la fonction cardiaque et les récepteurs à l’angiotensine II dans un modèle expérimental de rats avec induction d’une insuffisance cardiaque par l’isoprotérénol.
Méthode
Les rats Wistar ont été divisés de façon randomisée en groupe témoin et en groupe insuffisance cardiaque. Le groupe insuffisance cardiaque a été divisé secondairement en sous-groupes : castration ; castration + substitution testostérone ; et castration sham . L’évaluation échocardiographique et hémodynamique de la fonction cardiaque a été effectuée. L’apoptose et la fibrose ont été déterminées en utilisant la transferase déoxyribonucléotide terminale : (TUNEL) et la coloration trichrome Masson respectivement. L’expression des ARM messagers des récepteurs de l’angiotensine II (AT1 et AT2) a été évaluée en utilisant les techniques de PCR sur la transcriptase-polymerase reverse , alors que l’immuomarquage Western a été utilisé pour évaluer les niveaux d’expression de la protéine Bcl-2.
Résultats
La castration augmente de façon significative l’apoptose des cardiomyocytes ainsi que la fibrose induit par l’isoprotérénol ( p < 0,05). L’expression des ARN messager du récepteur à l’angiotensine 2 AT2 dans le groupe castration est augmentée alors que l’expression de la protéine Bcl-2 est diminuée, comparativement au groupe castration + administration de testostérone ( p < 0,05).
Conclusion
Ces résultats suggèrent que la thérapie androgénique pourrait jouer un rôle important dans les modifications physiopathologiques observées dans l’insuffisance cardiaque et avoir des effets bénéfiques pour son traitement.
Background
Chronic HF is a major health problem throughout the world and a leading cause of morbidity and mortality . Sex differences exist when determining the cause of cardiovascular diseases. Most studies have examined these sex differences by focusing on the effects of oestrogen on cardiovascular function. However, numerous additional studies have indicated that androgens can also have an effect on cardiovascular function. Testosterone levels are decreased in men with HF, while testosterone replacement therapy has been associated with significant increases in cardiac output and improved functional capacity as well as reduced symptoms in men with HF . The therapeutic benefits of testosterone in those with chronic HF can be attributed to a number of factors. For example, testosterone has vasodilatory properties and acute administration has been shown to lower peripheral vascular resistance, reduce cardiac afterload and increase the cardiac index. In addition, testosterone can modulate immune responses and improve insulin resistance while also exerting its effects on coagulation, obesity, endothelial function and alterations in skeletal muscle . However, the exact mechanisms underlying these effects mediated by testosterone on HF remain unclear.
The RAAS is known to play an important role in the pathophysiology and development of HF. RASS activity is increased in patients with HF, which could lead to cardiac remodelling and sympathetic activation. Both angiotensin II receptor type 1 (AT1 receptor) and angiotensin II receptor type 2 (AT2 receptor) are expressed in the heart, with localization on cardiomyocytes . A number of studies have indicated that angiotensin II receptor expression is altered in the hearts of animal models with HF. Moreover, Nio et al. reported that expression of both AT1 and AT2 receptor mRNA was upregulated in the infarcted and non-infarcted portions of the LV following coronary ligation.
A number of interactions between androgens and RAAS have been shown to occur at several organ sites. Androgens and androgen receptor systems are known to exert protective effects on angiotensin II-induced vascular remodelling . Androgens have also been shown to affect AT1a receptor mRNA abundance in the abdominal but not thoracic aortas of male mice . Therefore, the purpose of the present study was to determine whether changes in the androgen environment could induce alterations in the expression and activity of heart angiotensin II receptors in HF. Furthermore, we proposed that these activities of angiotensin II receptors are involved in the development of fibrosis and apoptosis in cardiomyocytes in HF.
Methods
All procedures were performed and approved in accordance with the Institutional Animal Care and Use Committee at Harbin Medical University. Male Wistar rats were obtained from the Laboratory Animal Centre of Harbin Medical University (Harbin, China). All animals were maintained on standard rat chow and water ad libitum and were housed in the Laboratory Animal Centre under conditions of controlled ambient temperature (22–24 °C) with a light-dark cycle of 12 h.
Castration and hormone replacement
Seventy sexually mature male rats (7 weeks of age, 160–220 g) were used in this study. Ten rats were randomly selected for the control group. The remaining 60 were divided into three equal groups randomly: castration + placebo (Cas+P); sham castration (S-Cas); and castration + testosterone replacement (Cas+T); Surgeries were performed while the rats were maintained under pentobarbital sodium anaesthesia (60 mg/kg). The rats were placed in a supine position and the testes were removed via a midline incision in the lower abdominal wall. Sham-operated rats received a midline incision that was immediately closed. For rats receiving testosterone propionate, replacement was initiated on the first day following castration. Testosterone propionate (amino acids, P.F., Tianjin, China) was administered once daily (2 mg/kg subcutaneously) for 10 weeks. Placebo (saline) was administered at the same dosage. The dosage and duration of treatments were based upon previously described protocols and data from preliminary experiments in our laboratory .
Induction of heart failure
Two weeks postcastration, isoproterenol (Sigma-Aldrich, St. Louis, MO, USA) was administered (340 mg/kg/day, subcutaneously) on two consecutive days. Excluding controls, all rats received an injection of isoproterenol. Eight weeks later, the numbers in the four groups were: Cas+P ( n = 8); S-Cas ( n = 9); Cas+T ( n = 10); control group ( n = 10).
Echocardiography and haemodynamic measurements
Ten weeks postcastration and hormone replacement, transthoracic echocardiography was performed while the rats were maintained under pentobarbital sodium anaesthesia (60 mg/kg). Two-dimensional and M-mode images were obtained using a 10 Mhz transducer connected to an ultrasonic echocardiographic system (Acuson Sequoia 512; Siemens AG, Erlangen, Germany). Systolic and diastolic dimensions of the LV were obtained from the M-mode view. Ejection fraction was calculated as follows: ejection fraction (%) = [(LVDV−LVSV)/LVDV] × 100, where LVDV and LVSV are left ventricular end-diastolic and end-systolic volumes, respectively. All variables were measured in triplicate and averaged. The investigator who performed the echocardiograms was blinded to the treatment group.
Haemodynamic measurements were also obtained for the estimation of cardiac function. The rats were anaesthetized using pentobarbital sodium (60 mg/kg) and allowed to breathe room air spontaneously. A 2 Fr micromanometer-tipped catheter filled with heparinized saline was fed through the right carotid artery into the LV and connected to a pressure transducer (model SPR-407; Miller Instruments, Houston, TX, USA). During this procedure, LV systolic and diastolic pressures and maximal rates of rise and fall in LV pressure were recorded. Resting haemodynamic measurements were performed in triplicate at 5 min intervals and the average values of these triplicates were used for statistical analyses.
Tissue preparation and histological analysis
Following haemodynamic measurements, harvested cardiectomy was performed and the hearts were weighed. A small portion of the heart was snap frozen in liquid nitrogen and stored at −80 o C until biochemical and molecular analyses; a second portion of the heart was fixed in 4% paraformaldehyde overnight. Following fixation, samples were embedded in paraffin and 4 μm sections were cut and stained. In order to calculate the ratio of the interstitial fibrosis area in the LV, samples were stained with Masson’s Trichrome and 10 fields were selected randomly from five individual sections. The percentage of fibrotic tissue infiltration in the LV was calculated as follows: (fibrotic tissue area)/(fibrotic tissue area + myocyte area) × 100%.
Analysis of apoptosis
Cardiomyocyte apoptosis was measured using an in situ TUNEL assay. TUNEL staining was performed according to the manufacturer’s instructions in the In Situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland). In brief, the tissue sections were deparaffinized and rehydrated using xylene and graded ethanol dilutions with a final rinse in distilled water. The tissue sections were then pretreated with 20 mg/L proteinase K for 30 min and incubated in the TUNEL reaction mixture in a humidified cabinet for 60 min at 37 °C. The tissue was then washed in 0.1 M phosphate buffer saline in triplicate for 5 min and incubated in converter-POD for 30 min at 37 °C. Finally, the tissues were washed in 0.1 M phosphate buffer saline in triplicate for 5 min and stained with a diaminobenzidine-POD substrate (Boster, Wuhan, China). Tissue sections were examined with a microscope at × 400 magnification and at least 100 cells were counted in 10 evenly spaced fields. The percentage of apoptotic cells was identified as the apoptotic index. All measurements were performed in a blind manner.
Real-time reverse transcriptase-polymerase chain reaction
Total RNA was isolated by the TRIZOL technique (Takara, Otsu, Shiga, Japan) according to the manufacturer’s instructions. Samples were quantified with a spectrophotometer at 260 nm and integrity was determined using ethidium bromide agarose gel electrophoresis. Reverse transcription was performed using the PrimeScript™ reverse transcription reagent kit (Takara, Otsu, Shiga, Japan). To examine mRNA levels of the AT1 receptor (primer sequences 5′-CATCGTCCACCCAATGAAGTC-3′ and 5′-GGGAACAAGAAGCCCAGAAT-3′) and AT2 receptor (primer sequences 5′-CACAAACCGGCAGATAAGCA-3′ and 5′-CAGGTCCAAAG-AGCCAGTCATA-3′) quantitatively, real-time reverse transcriptase-polymerase chain reaction amplification was performed using a SYBR Premix Ex Taq™ (Takara, Otsu, Shiga, Japan). Expression of GAPDH (primer sequences 5′-CAACG-ACCCCTTCATTGACC-3′ and 5′-GACGCCAGTAGACTCCACGAC-3′) was measured as an internal control for sample variations in the reverse transcription reaction.
Western Blot analyses
Bcl-2 protein expression was measured using Western immunoblotting as previously described . In brief, cardiac tissues were scraped into 0.3 mL lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], 100 μg/mL phenylmethylsulphonyl fluoride and 30 μg/mL aprotinin) and incubated for 30 min on ice. Protein concentrations were measured using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Fifty micrograms of protein were mixed and boiled in SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 5 min and then separated on SDS-PAGE gels (Bio-Rad, Hercules, CA, USA). Separated proteins were transferred to a PVDF membrane and were probed with polyclonal anti-Bcl-2 (1:1000) and anti-GAPDH antibodies (1:1000) (Santa Cruz Biotechnology, CA, USA) to detect target protein expression. The relative amount of Bcl-2 was determined by densitometry using ImageQuant software, with protein levels of GAPDH used as an internal control.
Statistical analyses
Data are presented as mean ± standard error of the mean. After establishing homogeneity of variance and normal distribution, a one-way analysis of variance was performed to determine differences among groups. If the analysis of variance was determined to be significant, the Student-Newman-Keuls test was used for post hoc pairwise comparisons. Statistical significance was defined as P < 0.05. Statistical analyses of data were performed using SPSS statistical software (version 14.0).
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