Resistance exercise and testosterone treatment alters the proportion of numerical density of capillaries of the left ventricle of aging Wistar rats

Abstract

Changes in the heart compartments that leads to pathological cardiac hypertrophy can be related to testosterone reduction in aging males since heart cells are susceptible to androgens. Resistance exercise delays the changes of aging.

Aim: This study aimed to analyze alterations of the left ventricle of aged rats subjected to resistance exercise with administration of testosterone.

Methods: Wistar rats were divided into five groups: C Group (control), S Group (sedentary), ST Group (sedentary treated with testosterone), T Group (trained) and TT Group (trained and treated with testosterone), strength training protocol and testosterone treatment were 16 weeks long. All groups were sacrificed at 16 months except for C group, sacrificed at 13 months.

Results: There was no change in the weight of the heart or the left ventricle between the groups. ST group showed increase in Nv [cap] density of capillaries and collagen, with no differences in interstitial space. Both trained groups (T and TT) showed increase in the numerical density of capillaries (Nv [cap]) and in the interstitial space, with no changes in collagen.

Conclusion: Resistance exercise combined with testosterone triggered a response of compensatory adjustment in the increase of Nv [cap], collagen and interstitial space, increasing perfusion and nutrition to the heart.

• Aging and exercise
• heart capillaries
• testosterone supplementation

Introduction

Aging is a continuous process characterized by a progressive decline of the homeostatic control which begins at conception. It is a dynamic and progressive process in which changes occur, both morphological, functional, and biochemical and psychological which determine the progressive loss of the individual's ability to adapt to the environment, leading to greater vulnerability and greater incidence of pathological processes [1,2].

Anatomical changes are the most obvious signs of aging and are manifested in the first place [2]. The WHO classifies the elderly starting at 60 years old with most countries considering 65 years old, the beginning of the old age [3]. As for the animals, Wistar rats become mature at 5 or 6 weeks of age and after that, each month of life is equivalent to 2.5 years of a human life. Although we understand that 13–16 months does not mean exactly old, it means 54–66% of the animal lifespan when most of the aging processes are almost fully established [4].

Cardiovascular diseases are directly related to aging and are one of the major concerns of modern society [2]. The cardiac tissue is susceptible to the aging process in which remodeling occurs, causing an imbalance in the organ compartments. Cardiac remodeling is conceptualized as structural or biochemical changes of muscular, vascular and interstitial components of the myocardium. Cardiac remodeling may also be associated with physical exercise, keeping their compartments in balance [5].

Many studies indicate that physical exercise is associated with significant reductions in the incidence of cardiovascular diseases and can serve as a therapeutic modality in the prevention and prognosis of these diseases, especially in the elderly [6,7].

Like any other living tissue, the heart muscle needs a blood supply of its own in order to provide it with oxygen and remove waste products. As a consequence, the myocardium needs to maintain a balance in its compartments. The heart irrigation is called coronary circulation. A unique feature of the coronary arteries and coronary blood flow is the large amount of oxygen extracted by the myocardium, even when the person is at rest [8,9].

The increase in the number of mitochondria, the capillary neoformation and the normal activity of myosin ATPase observed in a hypertrophied myocardium by physical exercise, prevent the imbalance between oxygen consumption and supply, and the occurrence of ischemia, unlike observed in pathological hypertrophy [10].

During aging, besides tissue changes, there are also changes in hormonal levels In males, the decrease in testosterone levels stands out. According to Osterberg et al. [11], 20% of men aged 60 years suffer from hypogonadism. This late onset hypogonadism is also called testosterone deficiency syndrome (TDS) and has several elements that are related to heart problems, such as metabolic syndrome [12]. It is known that reposition of this hormone with caution and monitoring determines the increase in muscle tissue and, as a consequence, an increase in muscle strength, decreasing fat tissue and increasing libido.

Heart cells have specific androgen receptors and are potentially susceptible to the direct influence of androgens. Androgens have a powerful regulatory influence on the heart [13].

The purpose of this study was to analyze the numeric density of the capillaries (Nv [cap]) of the left ventricle of elderly rats subjected to an individualized program of resistance exercise with administration of testosterone propionate.

Methods

All experiments and procedures were approved by the Ethical Committee of São Judas Tadeu University under the protocol number 015/2006.

In order to carry out his study, 25 thirteen-month male Wistar rats were used. The division of groups was as follows: Control Group (C), with five adult rats, sacrificed at 13 months; Sedentary Group (S), with five rats sacrificed at 16 months; Sedentary Treated with Testosterone Propionate Group (ST), with five rats sacrificed at 16 months; Trained Group (T), with five rats subjected to a strength training program from 13 to 16 months; and Trained and Treated with Testosterone Propionate Group (TT), with five rats subjected to a strength training program from 13 to 16 months. C Group did not participate in the training, because the animals were sacrificed at 13 months. Although we understand that 13–16 months does not mean exactly old, it means 54–66% of the animal lifespan when most of the aging processes are almost fully established [4]. Furthermore, as the aging process does not stop until the animal death, we can assume that, unless there is some kind of intervention, the degenerative process will only grow.

All animals underwent a pre-adaptation to the training protocol and equipment during five days. The equipment used to carry out the strength training program with the animals was a vertical ladder made of wood with iron steps. The height of the equipment (ladder) is 110 cm (43.3 inches) with an inclination angle of 80°. The top of the equipment had a plastic box lined with newspaper for the accommodation of the animals in the interval between sets [14].

The training protocol was progressive with the load being adjusted every week. The load was composed of lead weights that were attached to their tails with a velcro tape. The animals were supposed to climb the ladder to reach the resting area at the top that was considered one repetition. The adaptation process was one week with six repetitions every day.

The animals of the S and ST groups climbed the ladder only once a day, five times a week, without load, until their sacrifice, in order to cause a stress similar to the trained groups. The training of the animals of the T and TT groups was of six continuous repetitions per day, five times a week for 16 weeks with a rest interval of 45 s between repetitions. The load increase was established from Heyward's proposal [15]. As the load was related to the weight of animals, every week all animals were weighed and their loads adjusted. C Group did not participate in training, because the animals were sacrificed at 13 months.

The hormone used was PERINON® (testosterone propionate, veterinary use) from Perini Laboratory (São José, Brazil), with 100 ml in vial containing 1 g of testosterone propionate and peanut oil q.s.p. 100 ml. The injected dose was calculated according to the weight of the animals in the same proportion as that used in humans. The prescribed dose for a 150-pound adult is 200 mg of testosterone propionate.

The dosing was administered three times a week. Injections were given throughout the training protocol, totaling 33 intramuscular injections.

At the defined age, the animals were anesthetized and sacrificed. Soon after the hearts were removed, the great base vessels near the organ were sectioned. Then the hearts were sectioned transversely at the level of the coronal sulcus. Myocardial samples were removed from the compact layer of the LV free wall and from the interventricular septum. The ortotrip technique was used in this procedure. Therefore, we got random and uniformly isotropic (AUI) cuts. Isotropic is the organ or tissue which has the same characteristics in all directions, so: isotropy = homogeneity. Thus, histological isotropic cuts are those in which the structure appears homogeneous, different from the anisotropic cuts, in which there is an orientation in the presentation of the material. Currently, the assessment of many stereological parameters needs AUI cuts as a requirement. The heart is an anisotropic organ. Ventricular fibers are arranged in spirals. To obtain AUI cuts, it is relied in the ortotrip method. The heart must be sectioned into two consecutive cuts. The first cut should be performed at an angle determined randomly (e.g. by lottery). Then, the cutting face should be supported and again the heart must also be cut following a random angle. Now, we can admit that the obtained fragments contain isotropic tissue. The procedure may be repeated several times if we want more security in cases in which the material is highly anisotropic [16]. Then fragments of myocardium were fixed in 10% buffered formalin and dehydrated in a increasing series of alcohols, they were cleared in xylene, embedded in paraffin, sectioned in 7 μm thick cuts and stained with picrosirius red for light microscopy analysis [17].

The blades stained with Picrosirus red were used to analyze the numerical density (Nv) of capillaries, i.e. the number of capillaries present in the photographic field of the optical microscopy.

For this parameter, determinations of capillary density (Nv [cap]) were performed by counting the number of capillaries present in the photographic field of optical microscopy. The capillaries which touched the lines to the right and upwards on the screen were disregarded, while those which touched the lines on the left and downward were considered for the purpose of counting.

The data were presented as average and standard deviation, and to detect differences between groups, the analysis of variance (ANOVA) was used, followed by Tukey's post-hoc test with a significance level of 5%. The data were analyzed with the statistical software SPSS, version 20.0 (IBM SPSS Statistics version 20.0).

Results

Our results show that testosterone treatment in aged rats can produce different outcomes depending on whether resistance exercise is associated with this treatment. Table 1 shows all the data needed to support our discussion. It starts with the weight of the animals and their hearts and goes to details of this organ in all experimental groups.

Table 1. Body weight (BW), heart weight (HW) and relative heart weight (HW/BW %) of C, S, T, ST and TT groups.

Groups	BW (g)	HW (g)	HW/BW (%)
(C) n = 4	541.5 ± 51.70	1.51 ± 0.09	0.28 ± 0.032
(S) n = 6	546.6 ± 42.15	1.74 ± 0.12	0.32 ± 0.034
(T) n = 6	543.0 ± 69.15	1.52 ± 0.12	0.28 ± 0.021
(ST) n = 5	517.5 ± 70.12	1.51 ± 0.19	0.29 ± 0.065
(TT) n = 6	485.8 ± 60.20	1.67 ± 0.25	0.34 ± 0.051

Results are means ± standard deviation, n = number of animals.

We found no difference in the weight of the heart between all groups. When we look at the left ventricle weight (Table 2), there is an increase in the sedentary aged group (sacrificed with 16 months). This increased weight is not seen in the other groups with the same age that were treated with testosterone, sedentary or exercised.

Table 2. Left ventricle weight (LVW) and relative left ventricle weight (LVW/HW %) of C, S, T, ST and TT groups.

Groups	LVW (g)	LVW/HW (%)
(C) n = 4	0.91 ± 0.06	60.42 ± 7.74
(S) n = 6	1.20 ± 0.11*	69.61 ± 8.03
(T) n = 6	1.03 ± 0.13	68.06 ± 4.21
(ST) n = 5	1.01 ± 0.08	68.15 ± 13.58
(TT) n = 6	1.10 ± 0.16	66.39 ± 6.68

Results are means ± standard deviation, n = number of animals.

*p < 0.05 versus C.

Table 3 shows an interesting picture of the heart physiology of all experimental groups. It shows that the number of capillaries is significantly different in all treated groups, whether treated with testosterone, exercise or both when compared to sedentary or control groups. It also show that our results from collagen and the size of the interstitial space match the profile described on the literature with an increase in the interstitial space for groups T and TT and a value for ST lower than that from S group. Also, we observed a decrease in the percentage of collagen in the same groups (T and TT) when compared to S group, with the highest value registered to the ST group. This information can provide us evidence to understand the changes promoted by the treatments given to the animals.

Table 3. Numerical density (NV) of the capillaries, collagen volume (Col %) and interstitial space (Int) of the left ventricle of C, S, T, ST and TT groups.

Groups	Nv (Md ± DP) NV	COL, %	Nv (Md ± DP) INT
Control (C)	5.75 ± 1.95	4.18 ± 1.64	3.25 ± 2.87
Sedentary (S)	5.58 ± 2.32	5.19 ± 2.67*	4.97 ± 4.21*
Trained (T)	8.15 ± 2.92†	4.61 ± 1.60*†	6.82 ± 4.22*
Testosterone  Sedentary (ST)	8.00 ± 2.89†	6.21 ± 1.83*†‡	4.10 ± 2.97*†‡
Testosterone  Trained (TT)	8.75 ± 2.93*†	4.10 ± 1.55†‡¶	6.73 ± 5.55*†

Results are means ± standard deviation, Nv = left ventricle weight, LVW/HW = ratio left ventricle weight/heart weight.

*p < 0.05 versus C; †p < 0.05 versus S; ‡p < 0.05 versus T; ¶p < 0.05 versus ST.

Discussion

Our study analyzed the effects of an exercise program and testosterone administration on the number of capillaries of the left ventricle of aged animals. Observing the weight data presented in Tables 1 and 2, we can see that both testosterone and exercise have effects on the left ventricle weight, which can be involved with the cardiac remodeling in process.

Looking further in Table 3, we can have a general idea of the left ventricle and three main components of its regular and physiological work. These results taken together, suggest that when exercise is added to testosterone supplementation, the remodeling process becomes more efficient.

The cardiac remodeling can be understood as structural or biochemical changes of the muscular, vascular or interstitial elements of the myocardium. These elements may change in parallel with organ function, keeping them constant. This invariability happens in the heart of athletes, despite the increase in myocardial mass [4].

In our study, the trained (T and TT) and sedentary treated with testosterone (ST) animals indicated a statistical difference in relation to the numerical density of capillaries when compared to sedentary animals which did not receive testosterone (C and S). This result probably occurred due to myocardial remodeling. Due to the long-term training, there is a significant increase in blood volume to meet the needs of the heart. Exercise training promotes a significant increase in ventricular volume and an improved myocardial contractility; this enables an increase of microvascularization. The maximum oxygen uptake (VO₂max) is the main indicator of these changes and is directly related to the microvascularization. The increase in microvascularization in the heart during physical training happens because of a mechanism called neoformation [9,18].

The ST group demonstrated no difference compared to the trained animals, despite the Nv being lower in this group (data can be observed in Table 3).

Tagarakis et al. [19] reveal in their work that athletes who use AAS have an increase in the heart's microvascularization, but this increase does not accompany the hypertrophy induced by physical training. This causes an imbalance between supply and the demand of oxygen in the myocardium, especially during aerobic physical training, becoming a side effect of AAS. In the hypertrophy without physical exercise mainly observed in diseases or senescence, the proliferation of capillaries does not occur. However, there is an increase in the vascular bed volume to maintain the balance between myocardial compartments. The coronary reserve is what determines how far the hypertrophy is physiological.

Coronary reserve is conceptualized as the difference between the base flow of the coronary arteries and the possible maximum flow that this circulation can offer before a myocardium ischemic event occurs [5]. When maximum reserve is used, the limit of physiologic hypertrophy will be reached. If the stimulus for hypertrophy persists, the fibers will not receive additional nutrients, and will enter into degeneration, being replaced by collagen, promoting an imbalance between compartments and decreasing the compliance of the heart. As a result, the hypertrophy takes pathological features in animals.

Animals from T and TT groups showed the same levels of collagen as that of C group (data presented in Table 3). The S and ST groups demonstrated an increase in collagen. The S group animals had increased collagen in relation to C group, due to aging [20]. The myocardium fibers have an increase in volume parallel to the amount of interstice until the maximum capacity of vascular dilatation. This is a limiting factor in the process of physiological hypertrophy of the myocardium. Up to this point, the collagen connected between fibers behaves in a physiological manner without interfering with the organ compliance. In pathological hypertrophy, the amount of fibers decreases, and the interstitial space increases, resulting in an imbalance between the three compartments.

According to Balaji et al. [21], increases in the interstitial space are related to increases in the risk of arrythmia, especially for aged animals. However, exercise promotes a beneficial and physiological increase in the interstitium, giving this volume increase some different features than those found in the aging process.

Different than our results, Fontana et al. [22] showed reduction in the interstitial space of trained animals, but these animals were LDL knockout, and their hearts naturally had structural problems. In our study, we believe that the interstitial increase promoted by exercise helped the left ventricle work.

This is the difference between physiological and pathological hypertrophy. Beyond that limit, the physiological hypertrophy would only continue if there was hyperplasia of cardiac myocytes with maintenance of physiological connections of collagen, keeping the balance between the elements [5].

There was no difference in the heart weight (Table 1) and in the left ventricle (Table 2) of the ST and TT groups; however, hypertrophy was recorded in both groups, with the ST group suggesting a pathological hypertrophy and the TT group a physiological hypertrophy. This indicates that physical exercise has a significant preservation effect of physiological characteristics on the size and function of the heart [23].

The increase in the number of mitochondria and neoformation of capillaries in left ventricular hypertrophy (LVH) caused by training prevent the imbalance between supply and consumption of oxygen and the occurrence of ischemia, unlike what was seen in pathological hypertrophy [9].

The LVH and the increase in Nv of capillaries of practitioners of physical exercises have different hypertrophy characteristics than those associated with senescence and pathological hypertrophy, because it is not accompanied by fibrosis and other structural changes which undermine and compromise the function [9].

The LVH of athletes is physiological until it reaches the maximum capillary volume, i.e. until the use of all coronary flow reserve. Beyond this limit, the LVH becomes pathological. The interstice can be attacked by inflammatory diseases, disproportionally increasing this compartment over others, increasing the distance between the capillaries and the myocardium fibers, making its nutrition and functionality difficult. With aging, the collagen tissue is deposited with scarring sequelae of this process, deforming the framework on which the muscle works, causing damages to this muscle; there is degeneration of fibers, with resulting reduction of the muscle compartment, besides the destruction of capillaries, exacerbating even more the imbalance between the compartments. Physical exercise has systemic actions which directly influence the organism, slowing the morphofunctional changes of aging.

We can see that exercise promotes a greater nutritional and oxidative need of the tissue, and both lead to the production of angiogenic factors [20]. For this reason, the numerical density of capillaries was higher in the groups which performed exercises (T and TT).

We observed that testosterone supplementation increases collagen and microvascularization, but does not accompany cardiac hypertrophy, causing pathological hypertrophy. The testosterone supplementation associated with physical exercise has a protective effect associated with that balance, because the animals in the TT group maintained the same levels of collagen of young animals (C group), generating greater cardiac compliance. These animals also demonstrated greater increases in microvascularization, thus generating greater nutrition and oxygenation of the organ, maintaining a balance of myocardium components and minimizing the morphofunctional changes associated with aging. These results are in accordance with the results showed by Rydlewska et al. [24] and Florvaag et al. [25], both papers show beneficial effects of testosterone treatment in cases of heart problems.

Therefore, we conclude that resistance exercise combined with testosterone propionate can trigger a response of compensatory adjustment in the increase of the numerical density of the capillaries, increasing perfusion and heart tissue nutrition.

The testosterone propionate administered to rats induces left ventricular remodeling due to changes in its components.

This suggests that testosterone supplementation combined with resistance physical exercise promotes positive morphofunctional changes in the components of the left ventricular myocardium maintaining the balance between the compartments.

Declaration of interest

The authors report no declarations of interest.