Testosterone-induced hypertrophy, fibrosis and apoptosis of cardiac cells – an ultrastructural and immunohistochemical study

Background

Testosterone is the main androgen hormone and exerts its physiological effects through a genomic mechanism that is mediated by the androgen receptor [1]. However, a non-genomic pathway has recently been suggested [2]. These non-genomic effects are considered to be rapid, in contrast to the genomic effects, and several possible pathways have been identified. The activation of myocardial inflammation signaling enzymes of the MAPK family, mediated by the androgen receptor, has been documented, but interestingly this activation is not related to the receptor’s transcriptional activity [3,4].

Testosterone and its related androgens has significant anabolic and androgenic effects. Since its molecular identification, testosterone has been used as a therapy for hormone replacement, infertility and libido dysfunction, as well as an anti-aging agent [5,6]. The abuse of androgens for improvement of performance has become a common, yet serious, condition that may have detrimental effects on the athletes. Testosterone abuse has been linked to an increase of cardiovascular adverse events such as significant cardiac hypertrophy and myocardial infarction, virilization of women, prostate hyperplasia in men [6,7], liver hypertrophy and hepatocellular adenomas or carcinomas [8–12], apoptotic death of myocardial [13,14] and neuronal cells [15]. On the other hand, recent investigations have shown that testosterone might have anti-ischemic effects [16,17] and possibly inhibits plaque development [18,19].

The aim of this study was to examine the effect of testosterone administration at the ultrastructural level of the rat myocardium, as well as to investigate whether testosterone abuse can induce apoptosis. We used high doses of testosterone enanthate for a long period of time mimicking androgen abuse. The hypothesis was that testosterone induces hypertrophy, fibrosis and apoptosis of cardiac cells.

Material and Methods

STATISTICAL ANALYSIS:

The quantitative evaluation was made from photomicrographs of the myocardial tissue samples from each group by 1 of the authors (V.P.) blinded to the experimental groups. The area of fibrosis was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the fibrosis ratio was calculated by dividing the area of fibrosis by the total myocardial area. The dimensions and areas of mitochondria were measured with the use of ImageJ and were expressed as mean ± SE for each animal group. The Mann-Whitney U-test was used for comparisons between the 2 groups. Data was analyzed using PASW Statistics 18, Release Version 18.0.0, 2009 (SPSS, Inc., Chicago, IL). A value of p<0.05 was considered statistically significant. Finally, a quantitative measurement of the immunohistochemical staining for caspase-3 was performed using the ImageJ software.

Results

OPTICAL STUDY:

We observed significant myocardial hypertrophy of rats that received testosterone (Figure 1), whereas the myocardium of control rats appeared normal, without signs of hypertrophy (Figure 2).

ELECTRON MICROSCOPY:

Significant alterations were observed in the myocardial cells and the endothelial cells of the heart capillaries. We found edematous mitochondria in the myocardial cells of the experimental group along with some degree of intracellular edema (Figure 3). On the other hand, the mitochondria of the control group appeared to be normal (Figure 4). In order to evaluate any differences quantitatively, we measured and analyzed the shape and size characteristics of mitochondria of length exceeding 1.0 μm. The maximum length found was 7.96 μm.

We measured and compared the area (A), the length (L), the width (W) of the equivalent (same area) ellipse W=A/(πL) and the elongation (L/W) of 219 mitochondria in the experimental group and 23 mitochondria in the control group. The results presented in Table 1, show that the area and the width of the mitochondria were larger for the experimental group, whereas the length and the elongation were larger for the control group. This shows that the mitochondria in the experimental group are larger and more rounded. It is indicative that the top 5 values of the area of the mitochondria in the experimental group (9.68–30.39 μm2) were all higher than the highest value of the corresponding area in the control group (7.67 μm2); however, the difference was non-significant (p=0.93), as were the differences in length (p=0.30) and width (p=0.42). On the other hand, the difference in elongation between the 2 groups was statistically significant (p=0.04).

There was also disarrangement of the sarcomeres, with disorganization of the myofibrils and the Z discus (Figure 5). Between the capillaries and the myocardial cells we observed collagen fibers, which are an indication of myocardial fibrosis (Figures 6, 7). The nuclei in some endothelial cells had irregular shape (Figure 7). Endothelial cytoplasmic foot processes were abundant (Figure 3). A large number of pinocytoplasmic cysts inside the endothelial cytoplasm was observed (Figures 5,8). The appearance of the myofibrils and the mitochondria was normal in the control rats (Figure 4).

IMMUNOHISTOCHEMICAL STUDY:

Myocardial immunohistochemical staining for caspase-3 was negative for the rats in group B (control group) (Figure 9). On the other hand, the myocardial tissue of the rats which received testosterone (group A) exhibited significantly positive staining for caspase-3, which indicates the detection of apoptosis (Figures 10, 11). The differences are presented in Figure 12, in the form of RGB color histograms, determined by the use of ImageJ software. To quantify the differences, we calculated the Delta-E (ΔE76), as defined by the International Commission on Illumination (CIE), after conversion of the mean RGB values of each image into L*, a* and b* values in the L*a*b* color space [20]. The values of ΔE76 between the control and experimental groups were found to be 63.0 and 74.3, for Figures 9, 10 and 9–11, respectively, and only 13.7 between Figures 10, 11, which correspond to experimental group images. A value of ΔE76 approximately equal to 2.3 corresponds to a JND (just noticeable difference).

MASSON’S TRICHROME STAINING FOR COLLAGEN FIBERS:

Masson trichrome staining showed traces of local collagen fibrils (connective tissue) among the cardiac cells in the control group. Figure 13A shows Masson’s trichrome staining of a representative heart tissue section from a rat of the control group. The amount of collagen fibrils in the experimental group that received testosterone was higher compared to the control group.

In sections of cardiac veins, thickening of the muscular tunic with many foamy cells was observed as well as many collagen fibrils in the outer vascular tunica, surrounded by thick adipose tissue (Figure 13D). The presence of adipose tissue was evident around the arterioles wall as well. Within the adipose tissue a number of collagen fibrils were observed. The control group did not show similar characteristics.

Discussion

Androgen abuse has been linked to several serious adverse cardiovascular events. Cardiac arrhythmias, QT dispersion, atrial fibrillation, myocardial infarction, heart failure and atherogenesis have all been linked to androgen abuse by athletes [14]. Melchert and Welder proposed 4 hypothetical models of anabolic-induced adverse cardiovascular effects [21,22]:

Our study shows that the administration of testosterone in high doses exerts toxic effects on the myocardial cells. This probably correlates with the direct myocardial injury model of the effects of androgen-anabolic steroids on the myocardial cells. The mitochondria are particularly damaged and appeared edematous, with diminished cristae. The morphometric approach showed that the mitochondria of the experimental group were larger and more rounded.

The contractile apparatus showed signs of deterioration, with disorganization of the sarcomeres and the Z discus. These observations have been reported earlier and have been characterized as typical for early heart failure [23,24].

Another interesting finding of this study, using the Masson’s trichrome staining, was the presence of collagen fibrils inside the myocardium and particularly between the capillaries and the myocardium cells. This perivascular myocardial fibrosis, together with the hypertrophy that was also induced, may be the cause of myocardial ischemia [25,26] as well as a substrate for arrhythmias [27,28]. The presence of collagen was not noted in the hearts of the control rats. The collagen may increase the stiffness of the myocardium and reduce its compliance. Crisostomo et al. [3] reported that acute exposure of hearts to testosterone significantly reduces the −dP/dt (a measure of cardiac compliance).

The myocardial hypertrophy observed in the present study and also in our previous experiments [29], as well as by other researchers [30,31], has a significant role in the testosterone-induced reduction of myocardial compliance. The hypertrophy of the myocardium correlates with the enhanced expression of the androgen receptor after testosterone administration [29]. Myocardial hypertrophy is also associated with myocardial dysfunction due to abnormal intracellular calcium cycling [32]. Abnormalities of the circulating levels of other hormones may induce adverse cardiovascular effects. Increased levels of the protein hormone leptin have been found to induce cardiac hypertrophy although diastolic dysfunction was not associated with leptin levels [33]. Another set of hormones, the thyroid hormones, do have an impact on the myocardial diastolic properties among other various cardiovascular effects [34].

An unusually large number of micropinocytic vesicles was observed in the cytoplasm of the endothelial cells. Although it is common for these vesicles to occur in capillary endothelial cells, especially in the striated muscles, the abundance of such vesicles is a sign of heightened pinocytic activity that permits the cell to receive substances through the cell membrane [35].

Apoptosis is the programmed cell death and is mediated by 2 pathways: the extrinsic death receptor signaling pathway and the intrinsic mitochondrial control pathway [36,37]. Caspases exert significant action in both pathways. Caspase-3 (CPP 32) is a member of the interleukin-1 beta-converting enzyme (ICE) family of mammalian proteases that specifically cleaves substrates at the C-terminal side of aspartic residues. Members of this family have been implicated in apoptosis, and caspase-3 acts as a control mediator of programmed cell death in mammalian cells. Caspase-3 is synthesized as an inactive 32kD proenzyme and is processed during apoptosis to its active form, which is responsible for the cleavage of poly (ADP-ribose) polymerase (PARP), actin and sterol regulatory element binding protein (SREDP) [38–40]. In the present study, testosterone overdosing significantly activated apoptosis, as was clearly seen by immunohistochemistry. The staining for caspase-3 was negative in the control rats. Although it has been reported that apoptosis activated by testosterone enanthate is due to the ester [21], experimental exposure of myocardial cells to enanthate alone did not activate apoptosis [14]. Apoptosis causes the loss of myocardial cells and ultimately the depression of myocardial performance. The abuse of androgen anabolic substances has been causally linked with sudden cardiac death, myocardial infarction, ventricular remodeling and cardiomyopathy. These events are related to the activation of apoptosis due to AASs abuse. Myocardial death without coronary vessel disease or atherosclerosis has also been attributed to the activation of apoptosis by AASs [41,42].

Conclusions

This study showed that testosterone abuse produces significant myocardial hypertrophy and fibrosis as well as of the myofibrils, the mitochondria and the capillaries. The activation of apoptosis was a significant finding that indicates the direct myocardial injury caused by testosterone. As testosterone is now used for a variety of possible indications such as hormone replacement in the elderly or as an antianginal in cardiac patients, more research is required to clarify the exact biochemical route of action as well as the correct dose to prevent adverse effects.