This Article Abstract Full Text (PDF) All Versions of this Article: 144/10/4478    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Callies, F. Articles by Bonz, A. W. Search for Related Content PubMed PubMed Citation Articles by Callies, F. Articles by Bonz, A. W.

Administration of Testosterone Is Associated with a Reduced Susceptibility to Myocardial Ischemia

Departments of Endocrinology (F.C., B.A.) and Cardiology (H.S., K.H., S.F., A.L., S.B., A.W.B.), Medical University Hospital Wuerzburg, 97080 Wuerzburg, Germany; and Clinic III for Internal Medicine (R.H.G.S., B.B.), Laboratory of Muscle Physiology and Molecular Cardiology, University of Cologne, 50931 Cologne, Germany

Address all correspondence and requests for reprints to: Frank Callies, M.D., Department of Endocrinology, Medical University Hospital, Josef-Schneider-Strasse 2, 97080 Wuerzburg, Germany. E-mail: Callies_F{at}klinik.uni-wuerzburg.de.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study investigated the impact of testosterone on myocardial ischemia-reperfusion injury and corresponding intracellular calcium ([Ca²⁺]_i) metabolism. Nonorchiectomized mature male Wistar rats were randomly assigned to placebo, a single dose of testosterone undecanoate, or 5-dihydrotestosterone. In a further series, orchiectomized rats were treated with placebo. After 2 wk of treatment, the hearts were removed and placed in a Langendorff setup. The isolated, buffer-perfused hearts were subjected to 30 min of no-flow ischemia and 30 min of reperfusion. Recovery of myocardial function was measured by analyzing pre- and postischemic left ventricular (LV) systolic/diastolic pressure and coronary perfusion pressure simultaneously, together with [Ca²⁺]_i handling (aequorin luminescence). Calcium regulatory proteins were analyzed by Western blotting. LV weight/body weight ratio was increased after administration of testosterone vs. orchectomized rats. The recovery of contractile function was improved in testosterone-treated rats: at the end of the reperfusion, LV systolic pressure was higher and end-diastolic pressure was lower in testosterone-treated rats. End-ischemic [Ca²⁺]_i and [Ca²⁺]_i overload upon reperfusion was significantly lower in testosterone vs. orchiectomized rats, too. However, levels of calcium regulatory proteins remained unaffected. In conclusion, administration of testosterone significantly improves recovery from global ischemia. These beneficial effects are associated with an attenuation of reperfusion induced [Ca²⁺]_i overload.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE INCIDENCE OF coronary heart disease in premenopausal women is much lower than in age-matched men. This significant gender difference has been related to a protective role of 17ß-estradiol (1). Studies in experimental animals have demonstrated multiple beneficial effects of estrogens on the cardiovascular system, including amelioration of ischemia- and reperfusion-induced myocardial injury (2). However, randomized placebo-controlled clinical trials have failed to find evidence for a protective role of exogenously administered estrogens, possibly because of procoagulatory and proinflammatory activity of estrogens (3, 4). In contrast, the role of androgens for cardiovascular health has been largely neglected.

Anabolic androgenic steroids have been associated with myocardial ischemia, sudden cardiac death, and hypertension in athletes (5), leading to the view that androgens are detrimental for the cardiovascular system. However, anabolic androgenic steroids consist of a variety of different steroids with differing pharmacological properties. Moreover, the incidence of cardiovascular morbidity associated with anabolic androgenic steroid use has been difficult to determine, because of the clandestine nature of steroid use in athletes. No clinical study has yet demonstrated a conclusive link between anabolic androgenic steroids and fatal cardiovascular events. Epidemiological data (6) and an intervention study (7) rather suggest either a neutral or a beneficial effect of natural circulating androgens on coronary heart disease in males. Testosterone in men declines with age, and supplementation of elderly men with testosterone has recently received considerable attention (8). Furthermore, the use of testosterone esters like testosterone enanthate in males induces transient supraphysiological androgen levels after im application. Thus, evaluation of the role of testosterone for cardiovascular health is of growing importance. The association of anabolic androgenic steroids with myocardial infarction and sudden cardiac death (5) suggests that androgens may negatively affect myocardial tolerance to ischemia. We, therefore, tested the effects of testosterone treatment on the ventricular dysfunction caused by global ischemia, followed by reperfusion in an isolated rat heart preparation. In addition, because intracellular free calcium overload is of major importance for ischemia-reperfusion injury (9), we used aequorin bioluminescence to assess intracellular calcium ([Ca²⁺]_i) handling during myocardial ischemia-reperfusion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal model
A total of 104 mature male Wistar rats at an age of 8–10 wk and a body weight (bw) of 180–200 g were obtained from Charles River Breeding Laboratories (Kisslegg, Germany). Animals received water and normal rat chow ad libitum. In two consecutive series, rats were randomly assigned to one of three experimental groups: I, nonorchiectomized and placebo (P) (n = 27); II, nonorchiectomized and a single dose of the long-lasting testosterone preparation [testosterone undecanoate (TUD), 500 mg/kg bw] im (n = 32); and III, orchiectomized and placebo (ORX+P) (n = 30). Treatment was begun 14 d before removal of the hearts. Because we wanted to find out whether favorable effects of the aromatizable testosterone ester (TUD) could be related to estrogen generation from TUD, we included in the second series a group receiving 5-dihydrotestosterone (DHT), a nonaromatizible androgen. Nonorchiectomized rats received a sc implantation of DHT (75 mg/pellet per 21-d release) (n = 15).

TUD was provided by Jenapharm GmbH & Co. KG (Jena, Germany). DHT time-release pellets were manufactured by Innovative Research of America (Sarasota, FL). TUD is available in ampoules containing 1000 mg of the ester in 4 ml of castor oil. As placebo we administered the pure castor oil without TUD and time-release pellets without any DHT, respectively. Dosages of TUD (10) and DHT (11) were chosen to achieve supraphysiological testosterone and DHT concentrations.

The studies were conducted according to the guidelines of the American Physiological Society Principles for Research Involving Animals and Human Beings.

Isolated heart
Isolated hearts were dissected and perfused retrogradely as described previously (12). The hearts were paced at 5 Hz, coronary flow was set to 12 ml/min·g heart weight except during no-flow ischemia, and temperature was controlled at 37.0 ± 0.3 C. Isovolumic left ventricular (LV) developed pressure (LVDP) and LV end-diastolic pressure (LVEDP) were measured at an intracardiac balloon volume of 50% of volume of maximal developed pressure (Vol_max) after a pressure volume curve was obtained (13). [Ca²⁺]_i was measured using the aequorin bioluminescence method. Light signals were quantified by means of recently derived formulas and a time-dependent normalization constant L_max(t) (Ref. 12).

LV pressure recording
LV pressure tracings were digitized with a 12-bit analog-digital converter (sampling rate, 1 kHz) and stored on magnetic disk. The digital signal of the LV pressure tracing was further analyzed using custom software to obtain the following parameters: LVDP, LV systolic pressure, LVEDP, and T90 (i.e. time from peak P_s to 90% of relaxation).

Analysis of calcium overload in the first minute of reperfusion
Analysis of [Ca²⁺]_i was performed as previously described (12). To analyze Ca²⁺ overload occurring in the first minute of reperfusion, the following parameters were used: [Ca²⁺]_endisch was end-ischemic [Ca²⁺]_i; peak was the initial peak of Ca²⁺ signal in the first minute of reperfusion, reflecting the first Ca²⁺ influx; Oscill_max was the maximum of the first 10 transients of Ca²⁺ oscillations in the first minute of reperfusion, reflecting the amount of Ca²⁺ released from the overloaded sarcoplasmatic reticulum during each oscillation; I_(0–60) was the time integral of the first 60 sec of reperfusion, respectively, normalized by L_max(t), a global Ca²⁺ overload index. The time integral for I_(0–60) was chosen according to preliminary experiments, demonstrating that the major part of the peak of the reperfusion-induced Ca²⁺ overload is over within 1 min and that no significant differences could be detected for light integral measurements of at least 1 min (12). End-ischemic calcium was calculated directly before onset of reperfusion.

Experimental protocol (aequorin experiments)
After the aequorin loading procedure, the temperature was increased to 37 C within 10 min, and the hearts were paced at 5 Hz. After steady-state conditions were reached, a pressure-volume relationship was obtained to determine the volume at peak developed pressure (Vol_max) as previously described (13). To achieve comparable loading conditions (i.e. balloon volumes) in hearts of different sizes, function was compared at 50% of Vol_max, the volume at maximum developed. LV volume was set to 50% of Vol_max and kept constant for the remainder of the experiment. After stabilization of mechanical function and aequorin light signals, 15 min later no-flow ischemia was initiated. Pacing was discontinued 5 min after the initiation of ischemia. Hearts were reperfused after 30 min of ischemia. In all hearts, transient ventricular fibrillation occurred. Pacing was reinitiated after stabilization of the cardiac rhythm approximately 5 min after spontaneous defibrillation. The hearts were reperfused for 30 min after spontaneous defibrillation.

Quantification of [Ca²⁺]_i
[Ca²⁺]_i was measured in all Langendorff preparations. However, to exclude artifacts (washout, leakage, bleaching, etc.) only experiments with excellent [Ca²⁺]_i signal throughout the experiment were selected for analysis (12).

Aequorin light signals were recorded using the four-channel recorder in parallel with the LV pressure and coronary perfusion pressure tracings and were digitized, stored, and analyzed as described above for the LV pressure tracings. Briefly, at the end of each experiment, the heart was perfused with a solution containing 20 mmol/liter Ca²⁺ and Triton X-100 to lyse the aequorin-loaded cells and expose all of the remaining aequorin to Ca²⁺. This resulted in an instantaneous burst of light, subsequently declining to baseline within 10–20 min. The area under the curve was integrated to obtain a value for the total amount of light (L_max) emitted from the aequorin loaded into the myocytes. The ratio of the light signal vs. L_max is the fractional luminescence, which was converted into [Ca²⁺]_i concentrations by the use of a calibration curve derived in vitro (14).

The wave-averaged signals were analyzed for peak systolic Ca²⁺ and diastolic Ca²⁺ parallel to the analysis of the mechanical parameters.

Calculation of _s, _d, and relative wall thickness
The _s, _d (i.e. peak systolic and end-diastolic circumferential wall stress, respectively), and relative wall thickness, assuming spherical isovolumic conditions, were calculated as previously described (13).

Analysis of calcium regulatory proteins [phospholamban/Na⁺/Ca²⁺-exchanger/ryanodine-receptor/sarcoplasmic Ca²⁺-ATPase (SERCA) 2a]
Tissue preparation.
Myocardial tissue, which was not treated with Triton X-100, was snap-frozen in liquid nitrogen immediately after explantation of the heart. Then, 1.22 ± 0.14 g myocardium from the free LV wall was powdered in liquid nitrogen and thawed on ice in three volumes of chilled preparation buffer [sucrose 300 mM, phenylmethy-sulfonylfluoride 1 mM, piperazine-N,N-bis(2-ethanesulfonic acid) 20 mM, EDTA 10 mM, NaH₂PO₄ 50 mM (pH 7.4)] as described by Münch et al. (15). Care was taken to dissect myocardial tissue from connective tissue, vessels, and epicardium. Samples were minced 3 x 30 sec at 4 C with an Ultra Turrax T8 (Janke & Kunkel KG, IKA-Werke, Staufen i. Breisgau, Germany) followed by homogenization with a glass-Teflon potter (B. Braun AG, Melsungen, Germany). The resulting homogenate was further diluted with the storage buffer containing sucrose 400 mM, HEPES 5 mM, Tris 5 mM, EDTA 10 mM, NaH₂PO₄ 50 mM (pH 7.2), 1:1 volumes; frozen in liquid nitrogen; and stored in -80 C until use in Western blots. The myocardial preparations (homogenates) were further diluted in the above storage buffer to a final protein concentration of 2500 µg/ml. Protein concentration was finally verified by Bradford’s assay.

Immunoblot analysis.
Proteins were immunoblotted according to Towbin et al. (16) with modifications as described (17). Then, 40 µg of protein homogenates were thawed on ice and suspended in buffer (Tris HCl 0.5 mM, glycerol 10%, sodium dodecyl sulfate 2%, 2-mercaptoethanol 5%, bromphenolblue 0.05%). Proteins were separated with discontinuous PAGE with 4% and 17% acrylamide and transferred to polyvinylidene difluoride (PVDF) membranes by wet blotting. Transfer efficiency was verified by total protein staining of the gels with Coomassie brilliant blue and by staining of the protein bands on PVDF membranes (Western blots) with Ponceau S solution (Sigma Chemical Co., St. Louis, MO; ready-to-use, 5 min). The blots were blocked in 5% low-fat milk and washed with Tris-buffered saline and Tris-buffered saline with Tween [150 mmol/liter NaCl, 10 mmol/liter Tris-HCl, 0.05% Tween 20 (pH 7.5)]. Membranes were incubated with antibodies against phospholamban (1:1000), SERCA 2a (1:1250), Na⁺/Ca²⁺-exchanger (1:1000), and ryanodine-receptor type 2 (monoclonal mouse anti-ryanodine-receptor antibody, IgG₁, 1:1000). Calsequestrin was detected for control (1:1250). After washing procedures, we exposed membranes to the secondary antibody, a peroxidase-conjugated antibody (1:2500). Proteins were detected by an enhanced chemoluminescence assay (ECL Kit, Amersham-Life Science, Buckinghamshire, UK), exposed to x-ray film (Kodak X-OMAT Engineering, Eastman Kodak Co., Rochester, NY), and evaluated densitometrically with a commercially available computer program (ImageQuant, Molecular Dynamics, Krefeld, Germany). Protein levels were normalized to overall cardiac protein recovered from the myocardium, as determined by Bradford’s assay, and expressed as densitometric units per microgram of protein. Three independent Western blots were performed for each antibody. The staining of the protein bands of the Ca²⁺-regulatory proteins on PVDF membranes (Western blots) with Ponceau S solution showed high transfer efficiency. The position of the corresponding protein bands was verified with two markers giving molecular weight.

Materials.
Salts were high grade and were purchased from Merck (Darmstadt, Germany); piperazine-N,N-bis(2-ethanesulfonic acid) and phenylmethy-sulfonylfluoride were from Sigma. The 30% acrylamide/bisacrylamide was from Bio-Rad (Hercules, CA). The primary antibody used for SERCA 2a detection was a mouse monoclonal IgG₁ antibody against canine SERCA 2a protein (Affinity BioReagents Inc., Golden, CO). The antiphospholamban antibody was a mouse monoclonal IgG antibody against canine phospholamban purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The antibody against calsequestrin was a polyclonal rabbit antibody against canine calsequestrin from Swant (Bellinzona, Switzerland). The antibody against ryanodine-receptor was a rabbit polyclonal antibody (Affinity BioReagents Inc.), and the antibody against the Na⁺/Ca²⁺-exchanger was a polyclonal rabbit anti-NCX antibody from Swant. Secondary antibodies were either monoclonal sheep antimouse IgG peroxidase conjugated or goat antirabbit IgG conjugated with peroxidase from Sigma Immunochemicals (St. Louis, MO).

Immunoassays
The blood samples were allowed to clot, and the sera were separated by centrifugation and stored at -20 C until assayed.

Serum testosterone was determined by a commercially available RIA (Diagnostic Products Corp., Los Angeles, CA) with a sensitivity of 0.06 ng/ml. The intra- and interassay variance ranged from 6–15% and 9–16%, respectively.

Serum DHT was also determined by a commercially available RIA (Diagnostic Systems Laboratories, Sinsheim, Germany) with a sensitivity of 0.004 ng/ml. The intra- and interassay variance ranged from 3–6% and 5–12%, respectively.

Statistical analysis
Data are reported as mean ± SEM. With four experimental groups, six statistical comparisons are conceivable. Testing for this high number of comparisons with multifactorial ANOVA would overcorrect significance levels. Therefore, we limited the statistical analysis to biologically meaningful comparisons. Comparison of variables between two groups was made by using the unpaired Student’s t test. Bonferroni’s correction for multiple comparisons was applied to yield a significance level of 0.05:5 = 0.01. Calculations were performed by a commercially available program, StatView SE + Graphics (Brainpower Inc., Calabasas, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal characteristics and hormone determination
The bw, LVw (LV weight)/bw ratio, and serum testosterone concentrations were determined 2 wk after single-dose treatment (Table 1). The bw was not significantly different between the treatment groups. LVw/bw ratio was significantly higher after TUD administration vs. ORX+P (P < 0.008). Serum DHT and testosterone levels were significantly lower in the ORX+P-treated animals and significantly higher in the DHT-/TUD-treated animals, respectively. Endogenous testosterone was suppressed in DHT-treated animals due to suppression of pituitary gonadotrophs.

Cardiac function during ischemia and reperfusion
After the beginning of no-flow ischemia, LVDP declined to zero within 5 min in all groups (Fig. 1). Ischemic contracture developed after approximately 5 min up to nearly 38 mm Hg. At the end of the ischemic period, resting pressure declined slightly to nearly 29 mm Hg in all groups. No significant differences in mechanical function or resting pressure, respectively, could be seen up to the end of ischemia between the treatment groups. On reperfusion, the contractile function was significantly improved in testosterone (i.e. TUD and DHT)-treated rats (vs. ORX+P); at the end of the reperfusion period, LVDP was higher (Fig. 1), whereas LVEDP was significantly reduced (Fig. 2). The improved mechanical function under testosterone treatment resulted in a significantly lower coronary perfusion pressure (CPP) vs. ORX+P/P (Fig. 3).

View larger version (25K): [in this window] [in a new window]   FIG. 1. LVDP preischemic, during ischemia, and reperfusion. (P, n = 27; TUD, n = 32; DHT, n = 15; ORX+P, n = 30).

View larger version (26K): [in this window] [in a new window]   FIG. 2. LVEDP preischemic, during ischemia, and reperfusion. (P, n = 27; TUD, n = 32; DHT, n = 15; ORX+P, n = 30).

View larger version (23K): [in this window] [in a new window]   FIG. 3. CPP preischemic, during ischemia, and reperfusion. (P, n = 27; TUD, n = 32; DHT, n = 15; ORX+P, n = 30).

View larger version (14K): [in this window] [in a new window]   FIG. 4. [Ca2+]i concentrations at the end of ischemia. [Ca2+]i was significantly elevated in orchiectomized rat hearts compared with TUD-treated rats. *, P < 0.01. (P, n = 27; TUD, n = 32; DHT, n = 15; ORX+P, n = 30).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Overload of [Ca2+]i (integral) as quantified during the first 60 sec after reperfusion. [Ca2+]i integral was significantly elevated in orchiectomized rat hearts compared with TUD-treated rats (P < 0.01). (P, n = 27; TUD, n = 32; DHT, n = 15; ORX+P, n = 30).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In our in vitro model, we demonstrated for the first time a reduced susceptibility to myocardial ischemia-reperfusion injury after the administration of long-lasting supraphysiological androgen preparations (i.e. TUD and DHT). The functional recovery was significantly improved, demonstrated by a significantly higher LVDP and lower LVEDP (vs. P and ORX+P). This effect can partly be explained by a lower end-ischemic [Ca²⁺]_i leading to decreased [Ca²⁺]_i overload in the postischemic period as well as a reduced systolic [Ca²⁺]_i oscillation. Accordingly, orchiectomy was associated with an impaired recovery from ischemia.

Two weeks after TUD administration, a significant cardiac hypertrophy (vs. ORX+P) could be observed, characterized by an increase of LVw/bw ratio. This TUD-induced hypertrophy had no negative effects on cardiac performance in the preischemic period.

Measurements of [Ca²⁺]_i during cardiac ischemia and reperfusion are of interest because of the potential role that changes in [Ca²⁺]_i may play in contractile failure and in the initiation of ischemic arrhythmias and cell damage (9). In our study, the recovery of contractile function was significantly improved in all androgen-treated rats (TUD and DHT vs. ORX+P); at the end of the reperfusion period LVDP was higher, whereas CPP and LVEDP were significantly reduced. Analysis of end-ischemic [Ca²⁺]_i signaling revealed significantly lower levels in the TUD group. In the ORX+P group, significantly higher systolic [Ca²⁺]_i oscillations were found, which might enhance further cell damage.

This protection from reperfusion injury by testosterone could not be explained by an influence on the expression of classical calcium-regulatory proteins. We could not observe quantitative differences in the protein levels of phospholamban, Na⁺/Ca²⁺-exchanger, ryanodine-receptor type 2, or SERCA 2a between groups. However, we cannot exclude differences in activity of these proteins [e.g. due to changes in phosphorylation status (18)]. Testosterone might also prolong intracellular acidification and might protect the myofilaments against reperfusion injury (19).

[Ca²⁺]_i overload during early reperfusion is one cofactor that is responsible for reperfusion injury. The ability of the myocardial cell to eliminate [Ca²⁺]_i overload to restore normal calcium homeostasis is related to the amount of [Ca²⁺]_i overload at the beginning of reperfusion (20). In our study [Ca²⁺]_i overload indices were elevated in ORX-treated vs. TUD-treated rat hearts. The increased [Ca²⁺]_endisch in the ORX+P group might have further impact on the calcium sensitivity of the myofilaments. Decreased responsiveness of the myofilaments without desensitization to calcium has been shown to occur during the reperfusion period; it impairs contractile performance, and was associated with a transient calcium overload after initiation of reperfusion (21). Calcium overload during reperfusion is able to activate a calcium-sensitive neutral protease (calpain), which may partially cleave the ryanodine receptor and reduce its ability to release sarcoplasmic reticulum calcium (22). This dose-dependent effect might lead to myofilamentary degradation and consecutive reduced calcium sensitivity. Restoration of myofibrillar integrity after reperfusion of the myocardium will last for more than the postobservation period of 60 min of our study. Summarizing, the level of increased end-ischemic calcium is able to initiate various effects that are associated with impaired functional recovery of the postischemic myocardium.

Previous studies revealed that administration of testosterone improves coronary vasomotion by NO-dependent (23) and independent ways (24). Intracoronary administration of testosterone was able to cause coronary dilatation in men with established coronary artery disease (25). Coronary relaxation was found to be present in estrogen as well as in testosterone-treated coronary arteries, with 17ß-estradiol being more potent than testosterone. Both sex hormones are acting by inhibiting the prostaglandin F₂- and depolarization-induced, calcium-mediated coronary contraction (26). The improved postischemic recovery in our study might be supported by an improved coronary vasomotion allowing increased coronary blood flow during reperfusion and, therefore, reduced myocardial reperfusion injury. In our study, the recovery from global ischemia was improved by both TUD and DHT, suggesting that it is related to the androgenic activity of both preparations. However, it seemed that TUD treatment was more effective than the DHT. This might be related to the additional aromatization of TUD to estrogens, which does not occur with DHT treatment. Nevertheless, the improved coronary perfusion pressure might also be a result of decreased postischemic diastolic pressure and thus decreased compression of the myocardium on the microvasculature.

However, because no direct measurements of coronary blood flow were performed in our study, only indirect evidence is given by a significantly increased CPP in the reperfusion period in P as well as in ORX+P rats, compared with TUD- or DHT-treated rats (Fig. 3). This suggests a decreased microcirculatory resistance by an androgen-induced dilatation of macro- and microvessels. The improved CPP may be a result of decreased postischemic diastolic pressure, and thus a decreased compression of the myocardium on the microvasculature is possible.

Furthermore, in a recent study we have shown that testosterone administration leads to an induction of cardiac IGF-I mRNA expression and its downstream target phosphorylated AKT Ser(473) (27). Interestingly, IGF-I is an important regulator of vascular function by stimulating NO release from cultured vascular endothelium (28). In addition, both IGF-I administration and phosphorylated AKT Ser(473) activation have been shown to protect against ischemic reperfusion damage in experimental animals by inhibiting apoptosis (29, 30). Thus, it seems possible that up-regulation of antiapoptotic pathways by androgens contributes to the protective effects of androgens observed in this study.

There are some limitations of our study. Because the hearts were either stored in liquid nitrogen at -70 C immediately after the end of the experiment for analysis of the calcium regulatory proteins or lysed with Triton X-100 for calibration of the [Ca²⁺]_i signal, no myocardium was harvested to perform histological analysis. Possible reperfusion-induced morphological alterations due to calcium-induced disruption of the myocytes could therefore not be analyzed.

In conclusion, testosterone administration significantly improved the functional recovery of the myocardium after global no-flow ischemia, which was correlated with a lower [Ca²⁺]_i overload phenomenon during reperfusion. This protective effect seems to be primarily androgen mediated.

	Footnotes

 
The work was supported by a grant from the Bundesministerium für Bildung und Forschung [IZKF Wuerzburg, Teilprojekt E2, no. 01KS 9603, Germany (to F.C. and B.A.)] and the Deutschen Forschungsgemeinschaft DFG [Sonderforschungs-bereich SFB 355 Pathophysiologie der Herzinsuffizienz, Teilprojekt B9 (to A.W.B.)].

Abbreviations: bw, Body weight; [Ca²⁺]_i, intracellular calcium; CPP, coronary perfusion pressure; DHT, 5-dihydrotestosterone; LV, left ventricular; LVDP, LV developed pressure (systolic pressure - diastolic pressure); LVEDP, LV end-diastolic pressure; LVw, LV weight; n.s., not significant; ORX, orchiectomized or orchiectomy; P, placebo; P_s, time from peak P_s to 90% of relaxation; PVDF, polyvinylidene difluoride; SERCA, sarcoplasmic Ca²⁺-ATPase; TUD, testosterone undecanoate; Vol_max, volume of maximal developed pressure.

Received January 13, 2003.

Accepted for publication July 1, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Collins P, Rosano GM, Jiang C, Lindsay D, Sarrel PM, Poole-Wilson PA 1993 Cardiovascular protection by oestrogen—a calcium antagonist effect? Lancet 341:1264–1265[CrossRef][Medline]
2. Delyani JA, Murohara T, Nossuli TO, Lefer AM 1996 Protection from myocardial reperfusion injury by acute administration of 17ß-estradiol. J Mol Cell Cardiol 28:1001–1008[CrossRef][Medline]
3. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E 1998 Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 280:605–613[Abstract/Free Full Text]
4. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J, Writing Group for the Women’s Health Initiative Investigators 2002 Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288:321–333[Abstract/Free Full Text]
5. Dickermann RD, Schaller F, Prather I, McConathy WJ 1995 Sudden cardiac death in a 20-year-old bodybuilder using anabolic steroids. Cardiology 86:172–173[Medline]
6. Barrett-Connor E, Khaw K-T 1988 Endogenous sex hormones and cardiovascular disease in men. Circulation 78:539–545[Abstract/Free Full Text]
7. Alexandersen P, Haarbo J, Christiansen C 1996 The relationship of natural androgens to coronary heart disease in males: review. Atherosclerosis 125:1–13[CrossRef][Medline]
8. Bhasin S, Bagatell CJ, Bremner WJ, Plymate SR, Tenover JL, Korenman SG, Nieschlag E 1998 Issues in testosterone replacement in older men. J Clin Endocrinol Metab 83:3435–3448[Free Full Text]
9. Murphy JG, Marsh JD, Smith TW 1987 The role of calcium in ischemic myocardial injury. Circ Res 75:15–24
10. Callies F, Kollenkirchen U, von zur Mühlen C, Tomaszewski M, Beer S, Allolio B 2003 Testosterone undecanoate: a useful tool for testosterone administration in rats. Exp Clin Endocrinol Diabetes 111:203–208[CrossRef][Medline]
11. Cowley Jr BD, Rupp JC, Muessel MJ, Gattone 2nd VH 1997 Gender and effect of gonadal hormones on the progression of inherited polycystic kidney disease in rats. Am J Kidney Dis 29:265–272[Medline]
12. Stromer H, de Groot MC, Horn M, Faul C, Leupold A, Morgan JP, Scholz W, Neubauer S 2000 Na⁺/H⁺ exchange inhibition with HOE642 improves postischemic recovery due to attenuation of Ca²⁺ overload and prolonged acidosis on reperfusion. Circulation 101:2749–2755[Abstract/Free Full Text]
13. Stromer H, Cittadini A, Szymanska G, Apstein CS, Morgan JP 1997 Validation of different methods to compare cardiac function in isolated hearts of varying sizes. Am J Physiol 272:H501–H510
14. Kihara Y, Grossman W, Morgan JP 1989 Direct measurements of changes in intracellular calcium transients during hypoxia, ischemia, and reperfusion of the intact mammalian heart. Circ Res 65:1029–1044[Abstract/Free Full Text]
15. Münch G, Bolck B, Hoischen S, Brixius K, Bloch W, Reuter H, Schwinger RH 1998 Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium. J Mol Med 76:434–441[CrossRef][Medline]
16. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract/Free Full Text]
17. Münch G, Bölck B, Sugaru A, Schwinger RHG 2000 Isoform expression of the sarcoplasmic reticulum Ca²⁺ release channel (ryanodine channel) in human myocardium. J Mol Med 78:352–360[CrossRef][Medline]
18. Vittone L, Mundina-Weilenmann C, Said M, Ferrero P, Mattiazzi A 2002 Time course and mechanisms of phosphorylation of phospholamban residues in ischemia-reperfused rat hearts. Dissociation of phospholamban phosphorylation pathways. J Mol Cell Cardiol 34:39–50[CrossRef][Medline]
19. Kitakaze M, Weisfeldt ML, Marban E 1988 Acidosis during early reperfusion prevents myocardial stunning in perfused hearts. J Clin Invest 82:920–927
20. Kusuoka H, Porterfield JK, Weisman HF, Weisfeldt ML, Marban E 1987 Pathophysiology and pathogenesis of stunned myocardium: depressed Ca2+-activation as a consequence of reperfusion-induced cellular calcium overload in ferret hearts. J Clin Invest 79:950–961
21. Carrozza Jr JP, Bentivegna LA, Williams CP, Kuntz RE, Grossman W, Morgan JP 1992 Decreased myofilament responsiveness in myocardial stunning follows transient calcium overload during ischemia and reperfusion. Circ Res 71:1334–1340[Abstract/Free Full Text]
22. Lokuta AJ, Meyres MB, Sander PR, Fishman GI, Valdivia HH 1997 Modulation of cardiac ryanodine receptors by sorcin. J Biol Chem 272:25333–25338[Abstract/Free Full Text]
23. Chou TM, Sudhir K, Hutchison SJ, Ko E, Amidon TM, Collins P, Chatterjee K 1996 Testosterone induces dilation of canine coronary conductance and resistance in arteries in vivo. Circulation 94:2614–2619[Abstract/Free Full Text]
24. Yue P, Chatterjee K, Beale C, Poole-Wilson PA, Collins P 1995 Testosterone relaxes rabbit coronary arteries and aorta. Circulation 91:1154–1160[Abstract/Free Full Text]
25. Webb CM, McNeill JG, Hayward CS 1999 Effects of testosterone on coronary vasomotor regulation in men with coronary artery disease. Circulation 100:1690–1699[Abstract/Free Full Text]
26. Murphy JG, Khalil RA 1999 Decreased [Ca2+]i during inhibition of coronary smooth muscle contraction by 17ß-estradiol, progesterone, and testosterone. J Pharmacol Exp Ther 291:44–52[Abstract/Free Full Text]
27. Nahrendorf M, Frantz S, Hu K, von zur Muhlen C, Tomaszewski M, Scheuermann H, Kaiser R, Jazbutyte V, Beer S, Bauer W, Neubauer S, Ertl G, Allolio B, Callies F 2003 Effect of testosterone on post-myocardial infarction remodeling and function. Cardiovasc Res 57:370–378[Abstract/Free Full Text]
28. Tsukahara H, Gordienko DV, Tonshoff B, Gelato MC, Goligorsky MS 1994 Direct demonstration of insulin-like growth factor-I-induced nitric oxide production by endothelial cells. Kidney Int 45:598–604[Medline]
29. Buerke M, Murohara T, Skurk C, Nuss C, Tomaselli K, Lefer AM 1995 Cardioprotective effects of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci USA 92:8031–8035[Abstract/Free Full Text]
30. Wolfman JC, Palmby T, Der CJ, Wolfman A 2002 Cellular N-Ras promotes cell survival by downregulation of jun N-terminal protein kinase and p38. Mol Cell Biol 22:1589–1606[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Kohno, N. Takahashi, T. Shinohara, T. Ooie, K. Yufu, M. Nakagawa, H. Yonemochi, M. Hara, T. Saikawa, and H. Yoshimatsu Receptor-Mediated Suppression of Cardiac Heat-Shock Protein 72 Expression by Testosterone in Male Rat Heart Endocrinology, July 1, 2007; 148(7): 3148 - 3155. [Abstract] [Full Text] [PDF]

				J. Liu, S. Tsang, and T. M. Wong Testosterone Is Required for Delayed Cardioprotection and Enhanced Heat Shock Protein 70 Expression Induced by Preconditioning Endocrinology, October 1, 2006; 147(10): 4569 - 4577. [Abstract] [Full Text] [PDF]

				K. J. Milne, D. B. Thorp, C. W. J. Melling, and E. G. Noble Castration inhibits exercise-induced accumulation of Hsp70 in male rodent hearts Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1610 - H1616. [Abstract] [Full Text] [PDF]

				J. M. Vicencio, C. Ibarra, M. Estrada, M. Chiong, D. Soto, V. Parra, G. Diaz-Araya, E. Jaimovich, and S. Lavandero Testosterone Induces an Intracellular Calcium Increase by a Nongenomic Mechanism in Cultured Rat Cardiac Myocytes Endocrinology, March 1, 2006; 147(3): 1386 - 1395. [Abstract] [Full Text] [PDF]

				M. Quinkler, I. J. Bujalska, K. Kaur, C. U. Onyimba, S. Buhner, B. Allolio, S. V. Hughes, M. Hewison, and P. M. Stewart Androgen Receptor-Mediated Regulation of the {alpha}-Subunit of the Epithelial Sodium Channel in Human Kidney Hypertension, October 1, 2005; 46(4): 787 - 798. [Abstract] [Full Text] [PDF]

				F. Er, G. Michels, N. Gassanov, F. Rivero, and U. C. Hoppe Testosterone Induces Cytoprotection by Activating ATP-Sensitive K+ Channels in the Cardiac Mitochondrial Inner Membrane Circulation, November 9, 2004; 110(19): 3100 - 3107. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 144/10/4478    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Callies, F. Articles by Bonz, A. W. Search for Related Content PubMed PubMed Citation Articles by Callies, F. Articles by Bonz, A. W.