| BackgroundIt is well known that premenopausal women have lower incidence of coronary heart disease than age-matched men. This significant gender difference has been attributed at least in part to negative effects of testosterone on the cardiovascular system. Endogenous anabolic androgenic steroids (AAS) have been used by athletes to improve performance by increasing muscle mass and strength. Among the numerous documented toxic and hormonal effects of AAS, attention has been recently focused especially on the cardiovascular effects.It is reported that there are increases in blood pressure and peripheral arterial resistance, and there are also effects on the heart muscle, primarily left ventricular hypertrophy with restricted diastolic function. Severe cardiac complications (heart failure, ventricular fibrillation, ventricular thromboses, myocardial infarction or sudden cardiac death) in individual strength athletes with acute AAS abuse have also been reported. However, AAS consist of a variety of different steroids with differing pharmacological properties. No clinical study has yet demonstrated a conclusive link between physiological testosterone and fatal cardiovascular events. Epidemiological data and an intervention study rather suggest either a neutral or a beneficial effect ofnatural circulating androgens on coronary heart disease in men. Testosterone replacement in orchidectomized rats improved the recovery of myocardial function after ischemia/reperfusion injury. These processes involve changes in gene-expression controlled by intracellular androgen receptor mediated pathways.Recent studies have, however, demonstrated alternative, rapid intracellular androgen receptor-independent mode of testosterone action. For example, administration of testosterone acutely induces vasodilatation in the systemic, coronary, and pulmonary vascular beds. Acting in this way, testosterone might increase myocardial tolerance to ischemia. Er et al. have recently shown that testosterone is directly cytoprotective in the myocardium, through activation of ATP-sensitive K+ channels in the mitochondrial inner membrane. It has been proposed that these rapid androgen actions may be exerted through membrane receptors that stimulate early intracellular signaling pathways through interaction with G proteins. Jose Miguel Vicencio found that in rat neonatal cardiac myocytes, testosterone increases intracellular Ca2+ levels by a non-genomic mechanism, which involves a membrane androgen receptor, a PTX sensitive G protein, PLC, IP3 and IP3R as signaling pathway. Androgens act acutely to stimulate contractility of cardiac myocytes independent of other factors which can influence myocyte function in vivo.In this study, we examined the chronic and acute effect of testosterone treatment on rat myocardium. Furthermore, we hypothesize that testosterone exert its acute cardioprotective effects by a mitochondrial pathway, specifically via inhibition of mPTP opening. Also we tested the role of mPTP modulators in the effect of testosterone.ObjectivesThe aim of the present study was to investigate the chronic and acute effect of androgen replacement therapy on rat hearts against ischemia/reperfusion injury. Eight weeks after gonadectomy, all the heart were mounted on a Langendorff apparatus to assess contractile function. In the isolated myocytes model, cell viability was measured by trypan blue exclusion and cell contraction were observed using vediotracking system. We hypothesize that mitochondrial channel or pores are involved in the mechanisms of acute cardioprotective effect of testosterone.Methods1. Gonadectomy model and hormonal replacement therapyFor the gonadectomized (GDX) group, the cauda epididymidis, caput epididymidis, vas deferens, and testis were extracted. The vas deferens were ligated bilaterally and the testes removed. The vas deferens were reinserted into the scrotal sac and underlying membrane. The replacement regimen of testosterone propionate (TP) to TP group and vehicle to GDX group was begun 1 week after gonadectomy, in order to provide sufficient period to clear the endogenous steroids.2. Isolated Perfused Heart PreparationAfter 8 weeks all the rats were anesthetized and the hearts were excised rapidly, placed in ice-cold Krebs-Henseleit buffer, mounted on a Langendorff apparatus. In the present study, the isolated heart was subjected to 30 min of ischemia followed by 120 min of reperfusion, which induced myocardial injury.3. Lactate dehydrogenase measurementTo assess the extent of myocardial tissue injury, the effluent from the isolated perfused heart was collected at 5 min of reperfusion and lactate dehydrogenase (LDH) was spectrophotometrically assayed. LDH activity was expressed as units per liter.4. Measurement of the Area of RiskFor determination of infarct size, the coronary artery was reoccluded at the end of reperfusion, and a solution with 2.5% Evans blue was perfused. Hearts were then frozen and cut into slices, which were then incubated in a sodium phosphate buffer containing 1% (w/v) 2, 3, 5-triphenyl-tetrazolium chloride for 15 min to visualize the unstained infarcted region. Infarct and risk zone areas were determined by planimetry. The infarct size was expressed as a percentage of the risk zone.5. Primary culture of neonatal rat cardiomyocytesCardiac ventricular myocytes were prepared from neonatal (1- to 2-d old) Sprague-Dawley rats as follows. Hearts were excised and the ventricles trisected andincubated with collagenase type II (0.05%) and pancreatin (0.1%). Cardiac myocytes were plated in DMEM culture media supplemented with 15% fetal bovine serum.6. Determination of MTT formazan formationTo determine the viability of cardiac myocytes, we carried out the MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay as follows. The MTT assay is based on the ability of mitochondria to reduce MTT (a yellow tetrazolium dye) to MTT formazan (a blue mitochondrial by-product), the reduction is mediated by mitochondrial dehydrogenases in living but not in dead cells. The amount of MTT formazan in DMSO was then measured at a wavelength of 570 nm, using a spectrophotometer.7. Preparation of isolated ventricular myocytesIsolated adult ventricular myocytes from male Sprague-Dawley rats were obtained by enzymatic dissociation. Trypan blue exclusion was used as an index of the viability of the ventricular myocytes.8. Measurement of the mitochondrial membrane potential and reactive oxygen species productionMitochondrial membrane potential (Δψm) was measured using the fluorescent dye tetramethyl-rhodamine ethyl ester (TMRE). TMRE fluorescence was excited at 514 nm and collected at 590 nm. Reactive oxygen species (ROS) production was measured by loading with fluorescent probe DCFH-DA. Levels of DCF in cell lysates or in solutions were measured using 488 nm excitation/530 nm emission settings.9. Measurement of myocyte contractionMyocytes were placed in a chamber mounted on an inverted microscope. The chamber was perfused continuously at 2 ml/min with KH buffer, with 1% BSA and a gas phase of 95% O2/5% CO2 at room temperature. A video-tracking system was used to measure the peak velocity of cell shortening (+dL/dtmax), the peak velocity of cell relengthening (-dL/dtmax), the amplitude of contraction (dL) and the end-diastolic length of the isolated myocytes.Results1. Chronic effect of testosterone propionate on rat body weight and heart against ischemia/reperfusion injuryTP replacement inhibited gonadectomy induced increase of body weights.Gonadectomy significantly increased the infarct size and LDH release at the time of 5 min reperfusion in the isolated perfused rat heart subjected to 30 min ischemia and 120 min reperfusion compared with untreated control hearts, and TP replacement attenuated the injury of gonadectomy.2. Testosterone treatment on cultured cell viabilityTestosterone also increases cell viability in primary cultured cardiomyocytes against H2O2-stress.3. Testosterone acutely prevents H2O2-induced loss of cardiomyocytes cell viabilityApplication of H2O2 induced a dose dependent viability loss in cardiomyocytes measured by trypan blue exclusion. Cell death was detected following application of H2O2 (10, 100, 1000 μmol/L) for 10 min. Treatment with 100 μmol/L H2O2 for 10 min has been shown to cause nearly 60% viability loss in cardiomyocytes.Pretreatment of cardiomyocytes with testosterone (T) (10-8, 10-7, 10-6, 10-5, 10-4 μmol/L) protected the cells from the toxicity of H2O2, while T at final concentration of 10-9 has no effect. It also showed that T had a dose-dependent cardioprotective effect.Application of 20 μmol/L Atr, a known opener of the mitochondrial permeability transition pore, for 20 min before H2O2-stress, significantly attenuated the effects of T on cell viability. Blockade of the mitoKATP channel with a selective inhibitor, 5-HD (100 μmol/L) for 10 min before H2O2-stress also abolished the effects of T on cell viability.4. Testosterone prevented H2O2 induced depolarization of mitochondrial membrane potentialWe observed that H2O2-stress significantly increased the fluorescence intensity, which indicated that the Δψm was depolarized. Pretreatment with T beforeH2O2-stress prevented the depolarization of Δψm, and co-treatment with Atr or 5-HD attenuated the effect of T.5. Testosterone inhibited H2O2 induced ROS productionWe observed that H2O2-stress significantly increased the fluorescence intensity, which indicated that ROS were producted largely. Pretreatment with T or CsA before H2O2-stress prevented the production of ROS, and co-treatment with Atr or 5-HD attenuated the effect of T.6. Effect of testosterone on contraction of isolated ventricular myocytes H2O2-stress (100 μmol/L) produced significant decreases of ±dL/dtmax and dL.T attenuated the alterations of cell contraction induced by H2O2-stress. Perfusion with 10-5 μmol/L T 10 min prior to H2O2-stress significantly attenuated the inhibitory effect of H2O2-stress on +dL/dtmax and dL, and on -dL/dtmax at 2,6,10 min afterH2O2-stress. Application of 20 μmol/L Atr, a known opener of the mitochondrial permeability transition pore, for 20 min before H2O2-stress, significantly attenuated the effects of T on +dL/dtmax, while it had effect on -dL/dtmax only at 6 min and on dL at 8,10 min.ConclusionsIn conclusion, this study shows the chronic cardioprotection of testosterone against ischemia/reperfusion injury and acute effect against H2O2-stress injury. Gonadectomy significantly increases the infarct size and LDH release at the time of 5 min reperfusion. Furthermore, the acute cardioprotective effect conferred by testosterone is mediated by inhibiting opening of the mitochondrial permeability transition pore and activating the mitoKATP channel.
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