MICROSCOPY RESEARCH AND TECHNIQUE 77:727–734 (2014)
Effects of Aerobic Training, Resistance Training, or Combined Resistance-Aerobic Training on the Left Ventricular Myocardium in a Rat Model ^ MONICA RODRIGUES DE SOUZA,1 LEO PIMENTA,2 TANIA CRISTINA PITHON-CURI,3 MARCO BUCCI,3 RENATA GABRIEL FONTINELE,4 AND ROMEU RODRIGUES DE SOUZA5* 1
Department of Anatomy, Uninove University, S~ ao Paulo, Brazil Human Movement Laboratory, S~ ao Judas Tadeu University, S~ ao Paulo, Brazil 3 Institute of Physical Activity Sciences and Sports, Cruzeiro do Sul University, S~ ao Paulo, Brazil 4 Department of Anatomy, Faculty of Veterinary Medicine, S~ ao Paulo University, S~ ao Paulo, Brazil 5 Department of Anatomy, S~ ao Paulo and University and Human Movement Laboratory, S~ ao Judas Tadeu University, S~ ao Paulo, Brazil 2
KEY WORDS
endurance training; strength training; concurrent training; heart; Rattus norvegicus
ABSTRACT This study follows the left ventricular (LV) hypertrophy in rats undergoing aerobic training alone (A), resistance training alone (R), or combined resistance and aerobic training (RA) (usually referred as concurrent training) program. A sedentary control group (C) was included. LV remodeling was evaluated using electron and light microscopy. The LV weight to body weight (LVW: BW) increased 11.4% in A group, 35% in the R group, and 18% in the RA group compared to the C group. The LV thickness increased 6% in the A group, 17% in the R group, and 10% in the RA group. The LV internal diameter increased 19% in the A group, 3% in the R group, and 8% in the RA group compared with the C group. The cross-sectional area of cardiomyocyte increased by 1% with the A group, 27% with R group, and 12% with RA training. The capillary density increased by 5.4% with A training, 11.0% with R training, and 7.7% with RA training compared with the C group. The volume fraction of interstitial collagen increased by 0.4% with training A, increased by 2.8% with R training, and 0.9% with RA training. In conclusion, except for the LV internal diameter, which increased more in the A group, the cardiac parameters increased more in the R group than in the other groups and in RA group than in A group. Collagen density increased from 5.4 6 0.8% in the C group to 5.8 6 0.6% in the A group (n. s.) (P > 0.05), to 8.2 6 0.7% in the R group (P < 0.05), and to 6.3 6 0.4% in the RA group (P < 0.05). These results demonstrate a significant increase for collagen content in the LV with R and RA exercise, but the increase was higher with R training alone than with RA training. Microsc. Res. Tech. 77:727–734, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION It is well known that both, A training alone, and R training alone improve the performance and increase the reserve capacity of the myocardium (Anversa et al., 1983; Camargo et al., 2008; Difee et al., 2001; Kemi et al., 2002; Kwak et al., 2006; Rosa et al., 2005; Thomas et al., 2000; Vaz da Silva et al., 2002; Vitartaite et al., 2004). R training is associated in humans and animals with a concentric form of non-pathologic LV hypertrophy characterized by augmented ventricular wall thickness without changes in cavity size (Barauna et al., 2007; Haykowsky et al., 2002; Pluim et al., 2000). An increase in cardiomyocyte cross-sectional area is observed at the microscopic level (Barauna et al., 2007; Grossman et al., 1975). The potential effects of R exercise on changes in myocardial collagen tissue are unknown. In contrast, A training leads to an eccentric form of non pathologic cardiac hypertrophy, which is primarily C V
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characterized by an increased left ventricular (LV) cavity dimension (and thus, LV mass) (Jin et al., 2000; Kemi et al., 2002; Morganroth et al., 1975), cardiomyocyte hypertrophy (Fenning et al., 2003; Medeiros et al., 2004; Mokelke et al., 1997; Moore et al., 1993, 1995; Palmer et al., 1998; Wislïff et al., 2002), increased arteriolar number and length (Brown and Hudlicka, 1999; Laughlin and Mcallister, 1992; Pinheiro et al., 2006; Tomanek, 1994), and decreased interstitial collagen tissue (Pinheiro et al., 2006). Combined A and R training, also known as concurrent training, has been used with positive outcomes for *Correspondence to: Romeu Rodrigues de Souza, Rua Afonso de Freitas, 451, Ap 122, S~ ao Paulo, SP, Brazil, 04006-052. E-mail: [email protected] Received 3 April 2014; accepted in revised form 8 June 2014 REVIEW EDITOR: Prof. George perry Contract grant sponsors: Fapesp, CNPq, and CAPES. DOI 10.1002/jemt.22394 Published online 20 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
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glycemic levels in diabetic patients (Cuff et al., 2003; Larose et al., 2010; Loimaala et al., 2009; Maiorana et al., 2000; Reid et al., 2010; Sigal et al., 2007; Tan et al., 2012) and to improve functional capacity and strength in patients with chronic heart failure (Maiorana et al., 2000). However, to the best of our knowledge, studies on the adaptation LV changes of combined RA training using an animal model has not been performed. We designed this study to determine the effects of aerobic and resistance training alone versus a sedentary control group, and the incremental effects of doing both types of exercise (combined exercise training) versus aerobic or resistance training alone, on LV cardiomyocyte area, density of capillaries, and interstitial collagen.
peritoneally) and then euthanized. The animals were heparinized prior to tissue fixation to optimize perfusion-fixation. The hearts were excised in diastole, and the myocardium was perfused through the aorta at a constant pressure of 80 mm Hg using 0.1 M cacodylate buffer (3 min) followed by 2.5% glutaraldehyde solution diluted in cacodylate buffer. Next, the LV, including the septum, was isolated and weighed. A perpendicular section to the long axis of the LV including the entire thickness of the LV wall was cut at the level of the papillary muscles. Microscopic determinations of the thickness of the LV wall and the LV internal diameter were made by measuring the widths of four uniformly spaced sites along the length of each section using a computerized program (Axio Vision, Carl Zeiss AG, Jena, Germany).
MATERIALS AND METHODS Exercise Training Twenty-eight 8-week-old male Wistar rats were housed in a temperature- and light-controlled room and fed ad libitum. They were randomly divided into four groups of seven, as follows: control (C), A training, R training, and RA training. The rats in the R and RA groups trained to climb a 1.1 m vertical (80 incline) ladder with weights tied to their tails (Hornberger and Farrar, 2004). They were trained once each day over 8 weeks and each training session consisted of six climbs. The weight that was carried by these rats during each session was progressively increased at the beginning of each week. Over the course of 8 weeks, the amount of weight carried by each rat was equivalent to 50% of its body weight (BW). The BW was measured at the end of each week and the new weight to be carried by the animal during the next week was adjusted according to its body weight (Heyward, 1998). After each R training session, the animals from the RA group were subjected to a moderate treadmill running program. They and the animals from the A group were trained to run 1 h/day for 5 days/week at 60% of the maximum effort test (MET) on the treadmill at 15% gradient. The MET was performed at baseline on a treadmill at a speed of 0.3 K/h. Every 4 min the belt speed was increased in the same proportion (0.3 km/h) (Fontinele et al., 2013; Silva et al., 1997). The MET was performed every 4 weeks by the C, A, and RA groups. After the MET, the A and RA animals were subjected to 8 weeks of treadmill training 5 days a week, with increasing velocity up to 60% of that achieved in the effort test. In the first week after the test, the animals ran for 30 min, increasing this time by 10 min each week until they reach 60 min in the fourth week, where at the end of it, another MET was performed to adjust the intensity of the training for the next 4 weeks. Rat in the C group was placed on the stationary treadmill daily for 10 min. Animal handling was approved by our University Ethics Committee in adherence to the International Guiding Principles for Biomedical Research Involving Animals.
Morphological and Quantitative Analysis The left ventricle was divided into slices that were approximately 3 mm wide and 5 mm long, and the slices were post-fixed in osmium tetroxide in sodium cacodylate buffer for 1 h. The tissue was dehydrated in graded alcohol solutions, embedded in Epon resin, and sectioned so that the cardiomyocytes of one half of the blocks were cut in cross-sections, while the cardiomyocytes of the other half were cut in longitudinal sections. Thin sections for transmission electron microscopy were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). The longitudinal sections were used to evaluate the fixation state of the myocardium by measuring the sarcomere length in the four groups. Two randomly chosen blocks from each ventricle in which the cardiomyocytes were cut in cross-section were used for the quantitative analysis. This process was researcher blinded. The ultra-thin sections were placed on a copper grid, and 10 randomly chosen fields per block (20 fields per LV) were selected for micrographs taken on a Jeol transmission electron microscope (Jeol, Tokyo, Japan). Each of the 280 electron micrographs was analyzed by the Axio Vision software program (Carl Zeiss, Jena, Germany) to determine the cardiomyocyte cross sectional area, the capillary volume density, and the collagen fibril diameter. The cardiomyocyte cross sectional area was determined in ten electron micrographs from each heart with final magnifications of 6003, obtained from regions where the cardiomyocytes were transversely sectioned. The capillary volume density was expressed as the fraction (%) of the total area (volume reference) occupied by capillaries. It was evaluated using a test system with 84 sampling points, which was displayed on a monitor and calibrated. The following formula was used: Vv[cap] 5 Pp[cap]/PT, where Pp[cap] is the number of points in contact with capillaries and PT is the total number of test-points (84) (Br€ uel et al., 2002; Wulfsohn et al., 2004). The volume density can be expressed in the range of 0–1, but it can also be expressed as a percentage (Ribeiro, 2006), taking 100% as the total number of test points (84) and calculating the percentage of capillaries viewed from the number of points in contact with capillaries. The collagen content was determined by light microscopy in samples of myocardial tissue dissected
Cardiac Hypertrophy At the end of the experiment, one day after the last training session, each animal was anesthetized with sodium pentobarbital (3 mg/100 g body weight, intra-
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The means were calculated and the data were tested for significance. Statistical Analysis All data were expressed as mean 6 standard deviation (SD). The data were tested for normal distribution using the Kolmogorov–Smirnov test. Differences among groups were assessed by one-way analysis of variance (ANOVA). When a significant difference was detected, comparisons were performed by Tukey’s post hoc test. P values less than 0.05 were considered statistically significant.
Fig. 1. Absolute weight lifted by rats in the C, A, R, and RA groups at the beginning, (week 0) and after 8 weeks of training *P < 0.05 compared with C, A, R, and RA at week 0 and with C, A, and RA at week 8. **P < 0.05 compared with C, A, R, and RA at week 0 and with C and A at week 8. One-way ANOVA followed by Tukey’s test was used for the statistical analysis.
TABLE 1. BW, LVW, LV wall thickness and LV internal diameter of the four studied groups of rats Parameter / Groups
c (n57)
A (n57)
R (n57)
RA (n57)
Body wt, g 314 6 11.2 296 6 10.6 306 6 24 325 6 37 LVW:BW mg/g 2.8 6 0.2 3.12 6 0.2a 3.8 6 0.3b 3.3 6 0.2c LV wall 2.43 6 0.04 2.53 6 0.12 2.84 6 0.06b 2.63 6 0.02c thickness, mm LV internal 6.4 6 0.06 7.6 6 0.08d 6.6 6 0.04 6.9 60.1c diameter, mm Values are the mean 6 SD. a P < 0.05 compared with C. b P < 0.01 compared with group C, and P < 0.05, compared with group A and RA. c P < 0.05 compared with C and A. d P < 0.05 compared with C and R (P < 0.05).
from the LV anterior wall that were fixed in 10% Bouin’s solution for 24 h, embedded in paraffin, and used to make tangential histological sections of 5 mm. The sections were stained using the Picrosirius technique (Junqueira et al., 1979) and examined under polarized light. When studied with this method, tissues containing collagen fibers show intensely birefringent thick and thin fibers (Junqueira et al., 1978). To determine the collagen content range in the four groups, histological sections were entered into a KS400 digital analysing computer program (Zeiss, Germany), which quantified the area percentage (of 12,000 mm2) of the collagen fibers. Three sections per heart were analyzed using a microscope equipped with appropriate polarization lenses. In each section, five randomized fields were selected, and the area percentage of collagen fibers was quantified in each field. The collagen fibril diameter was determined in ten electron micrographs of each heart at a final magnification of 130,0003 obtained from regions where the fibrils were transversely sectioned. The minimum diameter of each fibril was measured using the image analyzer program. By commencing at one corner of each field and radiating outward in an arc, the diameters of the fibrils present in the field were determined. Microscopy Research and Technique
RESULTS One Repetition Maximum Figure 1 shows the absolute weight that was lifted by the groups of rats during the repetition maximum test. The repetition maximum values for the C, A, R, and RA groups (810 6 10 g) were similar at the beginning (day 0) of the study. After 8 weeks, the load lifted by the animals in the C and A groups was similar to that lifted in the first test. The load lifted by the animals in the R group (1,110 6 100 g) was 27% higher than the load lifted in the first test and that lifted by the animals in the RA group was 12% higher than that the first test (in both cases, P < 0.05). In summary, after 8 weeks of training, the capacity to lifting load augmented significantly in R and RA groups but not in C and A groups. Body Weight and Cardiac Hypertrophy Results about the body weight and cardiac hypertrophy in C and trained animals are summarized in Table 1. No significant differences in body weight were observed among the four groups, at the end of the study (P > 0.05). The LVW: BW increased 11.4% in A group (P < 0.05), 35% in the R group (P < 0.01), and 18% in the RA group (P < 0.05) compared to the C group. The LV wall thickness increased 6% in the A group (n. s.) (P > 0.05), 17% in the R group (P < 0.05), and 10% in the RA group (P < 0.05) when compared to the C group. The LV internal diameter increased 19% in the A group (P < 0.05), 3% in the R group (n. s.) (P > 0.05), and 8% in the RA group (P < 0.05) compared with the C group. In summary, except for the LV internal diameter, the cardiac parameters were higher in the R group compared with the other groups. They were also higher in RA group compared with C and A groups. The LV internal diameter was higher in group A than in the other groups. Cross-sectional Area of Cardiomyocytes The cross-sectional area of cardiomyocyte increased 1% in the A group (n. s.) (P > 0.05), 27% in the R group (P < 0.01), and 12% in the RA group (P < 0.05), when compared to the C group (Figs. 2 and 3). In summary, after 8 weeks of exercise, the cross-sectional area of cardiomyocyte was higher in the R group than in C, A, and RA group and it was higher in RA group than in A and C groups. Myocardial Capillary Density Capillary density increased 5.4 6 0.6% in the A group (n. s.) (P > 0.05), 11 60.5% in the R group
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Fig. 4. Capillary density (%) in the control (C) AND trained (A, R, and RA) groups of rats. Values are the means 6 SD. *P < 0.05 compared with C, A, and RA. **P < 0.05 compared with C and A.
(P < 0.05), and 7.7 6 0.3% in the RA group (P < 0.05), compared with the C group (Fig. 4). In summary, capillary density increased more in R group than in RA group, and increased more in RA group than in group A, compared with C group.
Fig. 2. Low-power electron micrographs of transverse sections from the LV wall of the C, A, R, and RA groups of rats that were used to determine the cross-sectional area of cardiomyocytes (cm) and the capillary density. Capillaries are indicated by arrows. IT—Cardiac interstitium. Scale bar: 10 mm.
Fig. 3. Mean data showing the cardiomyocyte area (mM2) of rats from the groups C, A, R, and RA after 8 weeks exercise training. Values are the means 6 SD. *P < 0.05 compared with C and P < 0.01, compared with A and RA; **P < 0.05 compared with C and A.
Myocardial Collagen Content and Collagen Fibril Diameter Histological sections from the LV myocardium stained with Picrosirius showed that the amount of collagen fibers was higher in the R group compared with the C, A, and RA groups and that it seems to be higher in the RA group compared to the C, and A groups (Fig. 5). Analysis of electron micrographs of groups C and A with magnification of 130,0003 showed that the collagen fibers were composed primarily of small-diameter fibrils. In the hearts from the R and RA groups, both large-and small-diameter collagen fibrils were present but with a predominance of larger diameter fibrils (Fig. 6). Collagen density increased from 5.4 6 0.8% in the C group to 5.8 6 0.6% in the A group (n. s.) (P > 0.05), to 8.2 6 0.7% in the R group (P < 0.05), and to 6.3 6 0.4% in the RA group (P < 0.05). These results demonstrate a significant increase for collagen content in the LV with R and RA training, but the increase was higher with R training alone than with RA training (Figs. 5 and 7). Statistical analysis revealed that the collagen fibril diameter was the greatest in the hearts from the R group (81 6 8 nm) compared with those from the C group (54 6 7 nm), A group (58 6 5 nm), and RA group (63 6 6 nm) (in all cases, P < 0.05) and that it was greatest in the hearts from RA group compared with those from C and A group (P < 0.05) (Fig. 8). In summary, collagen fibril diameter increased more with R training followed by RA training. DISCUSSION The primary findings of this study were that in Wistar rat, A training, and combined RA training each improved LV parameters and that R training is superior to both types of training. The results show that values obtained for the LV weight, LV thickness and Microscopy Research and Technique
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Fig. 5. Picrosirius red-stained ventricular sections from C, A, R, and RA groups viewed under polarized light, that were used to show the LV interstitial collagen. Scale bar: 100 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
for the cardiomyocyte cross-sectional area, capillary density, and collagen density with R training were superior to that obtained with training A alone or RA training in comparison with the C group. The results obtained with RA training for the same parameters were superior to that of training A, compared with the C group. The values obtained for the LVW:BW and to LV internal diameter with training A were inferior to that obtained with training R and RA, in comparison with the C group. The increase in LV parameters by R training can be explained by the high intraventricular pressure that is needed to open the aortic valve. During the ejection phase, high intraventricular pressure leads to increase in myocardial wall stress, which is the major stimulus for cardiac hypertrophy in the pressure-overloaded heart (Kasikcioglu, 2004). Microscopy Research and Technique
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Fig. 6. High-power electron micrographs of transverse-sectioned collagen fibers from the LV wall of the C, A, R, and RA groups of rats. The electron micrographs are viewed with the same magnification showing collagen fibrils of large (*) and short diameter (arrows). Scale bar: 100 nm.
Conversely, during A training, in addition to the increased cardiac output, the blood pressure increases, although it does not increase to the same extent as during R training. Consequently, the aerobically trained LV must adapt to both volume and pressure loads (Mihl et al., 2008). The heart responds by increasing the LV internal diameter (Fenning et al., 2003; Mokelke et al., 1997; Moore et al., 1993, 1995; Moore and Palmer, 1999; Palmer et al., 1998; Pluim et al., 2000). Consequently, during A training, the volume load is the prevailing factor acting on the ventricular wall; therefore, the aerobic-trained heart develops eccentric hypertrophy (Vinereanu et al., 2002). The reason why RA induced a specific form of cardiac hypertrophy is not known. It seems that in this
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Fig. 7. Mean data for the collagen density (%) in the LV from C, A, RA, and R groups of rats. Values are the means 6 SD. *P < 0.05 compared with C, A, and RA. **P < 0.05 compared with C and A.
Fig. 8. Mean data for collagen fibril (CF) diameter in the LV from C, A, R, and RA groups of rats. Values are the mean 6 SD. *P < 0.05 compared with C, A, and RA. **P < 0.05 compared with C and A.
form of training the cardiac hypertrophy produced by R training was affected by training A. Can A training inhibit protein synthesis? A possibility is that A training limits signaling for muscle growth. However, further studies need to be done to resolve this issue. This study demonstrated that animals receiving each type of exercise training showed different influence in the cross-sectional area of cardiomyocyte. The increasing in cardiomyocyte area in response to R training, results from a pressure overload (increased post-load) on the heart (Fleck, 1988; MacDougall et al., 1992). This pressure overload leads to a higher final systolic pressure to create a parallel response in the sarcomeres and to augmented LV wall thickness that characterizes the concentric hypertrophy (Effron, 1989; Shapiro, 1984). The expansion of cross-sectional area of cardiomyocyte is caused by a parallel sarcomere replication, which can contribute to the enhancement of the intrinsic contractility of these cells (Perrault and Turcotte, 1994). In addition, the intrinsic contractility of the cardiomyocyte may be a potential mechanism underlying the training-induced improvement of myocardial contractile function. Therefore, the
architecture of heart that is subjected to volumetric overload is altered via hypertrophy, promoting LV remodeling to normalize the parietal tension in line with Laplace’s Law (Lorell and Carabello, 2000; Zhang et al., 2011). Conversely, the A training increases the venous return and, consequently, imposes a volumetric overload (increased pre-load) on the heart. This overload results in the elevation of the final diastolic pressure experienced by the heart to produce a response in the sarcomeres, increasing the length of the cardiomyocyte and the size of the LV cavity, which characterizes eccentric hypertrophy (Mokelke et al., 1997; Moore et al., 1993; Palmer et al., 1998, 1999). In this study, the myocardial capillary volume density was altered by both R and RA training. Notably, the capillary volume density was most increased following R training alone. The capillary volume density in the RA and R groups may have increased due to the neo-formation mechanism (angiogenesis) in agreement with previous results following A training (Amaral et al., 2000). The neo-formation of vessels occurs in animals with impaired myocardium and in healthy animals to improve the blood supply to the myocardium (Brown and Hudlicka, 1999; Laughlin and Mcallister, 1992; Tomanek, 1994). This study demonstrated that the interstitial collagen content and composition were influenced differently by each type of training indicating that the myocardial collagen is likely remodeled differently by different types of training. One possible explanation is that the myocardial collagen degradation is related to cardiac pressure overload. In fact, the regulation of fibril collagen mRNA levels following pressure overload may be a result of hemodynamic changes and their impact on cardiac fibroblasts (Eghbali, 1990). Transforming growth factor-b1 (TGF-b1), which is present in cardiac fibroblasts, stimulates collagen gene expression in the extracellular matrix (Eghbali et al., 1989). Therefore, TGF-b1 expression may increase with pressure overload, which could explain the increase in collagen transcription in the R and RA exercised heart. However, additional studies are needed to determine the mechanisms by which the training affects myocardial collagen. Our trial was not designed to study effects of exercise volume or duration per se, and the superior effect of R training in some cardiac parameters may reflect the greater effect of training performed by the R group. Furthermore, because the physiologic effects of RA training differ from those of the A training alone, we cannot assume that our results reflect only additional training time. If our findings simply reflected duration of active training, we would expect that the effect of A training and of R training alone on myocardial parameters would be less than half that of RA training (Sigal et al., 2007). Instead, the effects of the A training and RA training on the myocardium were quite similar, and those of R training were twice those of A training. CONCLUSION This data suggest that RA training was more effective than training A, in eliciting improvements on the LVW: BW, LV wall thickness, and cross-sectional area of cardiomyocyte, capillary density, and collagen fibril Microscopy Research and Technique
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diameter and volume fraction of cardiac interstitial collagen. The LV internal diameter was better improved by training A. The results of this study also indicate that R training was more effective than RA training and A training in eliciting improvements on such parameters. Further studies are needed to evaluate the effect of RA training versus A and R training on other cardiac parameters under different conditions. REFERENCES Amaral SL, Zorn TM, Michelini LC. 2000. Exercise training normalizes wall-to-lumen ratio of the gracilis muscle arterioles and reduces pressure in spontaneously hypertensive rats. J Hypertens 18:1563–1572. Anversa P, Levicky V, Beghi C, McDonald SL, Kikkawa Y. 1983. Morphometry of exercise-induced right ventricular hypertrophy in the rat. Circ Res 52:57–64. Barauna VG, Rosa KT, Irigoyen MC, de Oliveira EM. 2007. Effects of resistance training on ventricular function and hypertrophy in a rat model. Clin Med Res 5:114–120. Brown MD, Hudlicka O. 1999. Exercise, training and coronary angiogenesis. Adv Organ Biol 7:155–196. Br€ uel A, Oxlund H, Nyengaard JR. 2002. Growth hormone increases the total number of myocyte nuclei in the left ventricle of adult rats. Growth Horm IGF Res 15:256–264. Camargo MD, Stein R, Ribeiro JP, Schvartzman PR, Rizzatti MO, Schaan BD. 2008. Circuit weight training and cardiac morphology: a trial with magnetic resonance imaging. Br J Sports Med 42:141– 145. Cuff DJ, Meneilly GS, Martin A, Ignaszewski A, Tildesley HD, Frohlich JJ. 2003. Effective exercise modality to reduce insulin resistance in women with type 2 diabetes. Diabetes Care 26:2977– 2982. Diffee GM, Seversen EA, Titus MM. 2001. Exercise training increase the Ca(21) sensitivity of tension in the rat cardiac myocytes. J Appl Physiol 91:309–315. Effron MB. 1989. Effects of resistive training on left ventricular function. Med Sci Sports Exerc 21:694–697. Eghbali M, Blumenfeld OO, Seifter S, Buttrick PM, Leinwand LA, Robinson TF, Zern MA, Giambrone MA. 1989. Localization of types I, III and IV collagen mRNAs in rat heart cells by in situ hybridization. J Mol Cell Cardiol 21:103–113. Eghbali M, Weber KT. 1990. Collagen and the myocardium: fibrillar structure, biosynthesis and degradation in relation to hypertrophy and its regression. Mol Cell Biochem 96:1–14. Fenning A, Harrison G, Dwyer D, Rose Meyer R, Brown L. 2003. Cardiac adaptation to endurance exercise in rats. Mol Cell Biochem 251:51–59. Fleck SJ. 1988. Cardiovascular adaptations to resistance training. Med Sci Sports Exerc 20:S146–S151. Fontinele RG, Mariotti VB, Vazzoler e AM, Ferr~ ao JS, Kfoury JR, Jr, De Souza RR. 2013. Menopause, exercise, and knee. What happens? Microsc Res Tech 76:381–387. Grossman W, Jones D, McLaurin LP. 1975. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest 56:56–64. Haykowsky M, Dressendorfer R, Taylor D, Mandic S, Humen D. 2002. Resistance-training and cardiac hypertrophy: Unravelling the training effect. Sports Med 32:837–849. Heyward VH. 1998. Designing resistance training programs. In: Vivian HH, editor. Advanced fitness assessment and exercise prescription, 3rd ed. Human Kinetics Publishers. Champaign, Illinois. pp. 121–144. Hornberger TA, Farrar RP. 2004. Physiological hypertrophy of the FHL muscle following 8 weeks of progressive resistance exercise in the rat. Can J Appl Physiol 29:16–31. Jin H, Yang R, Li W, Lu H, Rayan AM, Ogasawara AK, Van Peborgh J, Paoni NF. 2000. Effects of exercise training on the cardiac function, gene expression, and apoptosis in rat. Am J Physiol Heart Circul Physiol 279:H2994–H3002. Junqueira LC, Cossermelli W, Brentani R. 1978. Differential staining of collagens type I, II and III by Sirius Red and polarization microscopy. Arch Histol Jap 41:267–274. Junqueira LC, Bignolas G, Brentani. 1979. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histoch J 11:447–455.
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Shapiro LM. 1984. Physiological left ventricular hypertrophy. Br Heart J 52:130–135. Sigal RJ, Kenny GP, Boule NG. Wells GA, Prud’homme D, Fortier M, Reid RD, Tulloch H, Coyle D, Phillips P, Jennings A, Jaffey J. 2007. Effects of aerobic training, resistance training, or both on glycemic control in type 2 diabetes. A randomized trial. Ann Intern Med 147: 357–369. Silva GJJ, Brum PC, Negr~ ao CE, Kriger EM. 1997. Acute and chronic effects of exercise on baroreflexes in spontaneoously hypertensive rats. Hipertension 30:714–726. Tan S, Li W, Wang J. 2012. Effects of six months of combined aerobic and resistance training for elderly patients with a long history of type 2 diabetes. J Sports Sci Med 11:495–501. Thomas DP, Zimmerman SD, Hansen TR, Martin DT, McCormick RJ. 2000. Collagen gene expression in rat left ventricle: Interactive effect of age and exercise training. J Appl Physiol 89:1462–1468. Tomanek RJ. 1994. Exercise-induced coronary angiogenesis: A review. Med Sci Sports Exerc 26:1245–1251. Vaz da Silva MS, Boochi EA, Guimar~ aes GV, Padovani CR, da Silva MHG, Pereira SF, Fontes RD. 2002. Benefits of exercise training in the treatment of heart failure. Study with a control group. Arq Brasil Cardiol 79:357–362.
Vinereanu D, Florescu N, Sculthorpe N, Tweddel AC, Stephens, M.R, Fraser AG. 2002. Left ventricular long-axis diastolic function is augmented in the hearts of endurance-trained compared with strength-trained athletes. Clin Sci (Lond) 103:249–257. Vitartait e A, Vainoras A, Sedekerskiene V, Poderys J. 2004. The influence of aerobics exercise to cardiovascular functional parameters of 30–40 year old women. Medicina (Kaunas) 40:451–458. Watson ML. 1958. Staining of tissue sections for electron microscopy with heavy metals. J Biophysic Biochem Cytol 25:475–478. Wislïff U, Loennechen JP, Currie S, Smith GL, Ellingsen Ø. 2002. Aerobic exercise reduces cardiomyocyte hypertrophy and increases contractility, Ca21, sensitivity and SERCA-2 in rat after myocardial infarction. Cardiovasc Res 54:162–174. Wulfsohn D, Gundersen HJ, Vedel Jensen EB, Nyengaard JR. 2004. Volume estimation from projections. J Microsc 215:111–120. Zhang Z, Tendulkar A, Sun K, Saloner DA, Wallace AW, Ge L, Guccione JM, Ratcliffe MB. 2011. Comparison of the YoungLaplace law and finite element based calculation of ventricular wall stress: Implications for post infarct and surgical ventricular remodeling. Ann Thorac Surg 91:150–156.
Microscopy Research and Technique
Effects of Aerobic Training, Resistance Training, or Combined Resistance-Aerobic Training on the Left Ventricular Myocardium in a Rat Model ^ MONICA RODRIGUES DE SOUZA,1 LEO PIMENTA,2 TANIA CRISTINA PITHON-CURI,3 MARCO BUCCI,3 RENATA GABRIEL FONTINELE,4 AND ROMEU RODRIGUES DE SOUZA5* 1
Department of Anatomy, Uninove University, S~ ao Paulo, Brazil Human Movement Laboratory, S~ ao Judas Tadeu University, S~ ao Paulo, Brazil 3 Institute of Physical Activity Sciences and Sports, Cruzeiro do Sul University, S~ ao Paulo, Brazil 4 Department of Anatomy, Faculty of Veterinary Medicine, S~ ao Paulo University, S~ ao Paulo, Brazil 5 Department of Anatomy, S~ ao Paulo and University and Human Movement Laboratory, S~ ao Judas Tadeu University, S~ ao Paulo, Brazil 2
KEY WORDS
endurance training; strength training; concurrent training; heart; Rattus norvegicus
ABSTRACT This study follows the left ventricular (LV) hypertrophy in rats undergoing aerobic training alone (A), resistance training alone (R), or combined resistance and aerobic training (RA) (usually referred as concurrent training) program. A sedentary control group (C) was included. LV remodeling was evaluated using electron and light microscopy. The LV weight to body weight (LVW: BW) increased 11.4% in A group, 35% in the R group, and 18% in the RA group compared to the C group. The LV thickness increased 6% in the A group, 17% in the R group, and 10% in the RA group. The LV internal diameter increased 19% in the A group, 3% in the R group, and 8% in the RA group compared with the C group. The cross-sectional area of cardiomyocyte increased by 1% with the A group, 27% with R group, and 12% with RA training. The capillary density increased by 5.4% with A training, 11.0% with R training, and 7.7% with RA training compared with the C group. The volume fraction of interstitial collagen increased by 0.4% with training A, increased by 2.8% with R training, and 0.9% with RA training. In conclusion, except for the LV internal diameter, which increased more in the A group, the cardiac parameters increased more in the R group than in the other groups and in RA group than in A group. Collagen density increased from 5.4 6 0.8% in the C group to 5.8 6 0.6% in the A group (n. s.) (P > 0.05), to 8.2 6 0.7% in the R group (P < 0.05), and to 6.3 6 0.4% in the RA group (P < 0.05). These results demonstrate a significant increase for collagen content in the LV with R and RA exercise, but the increase was higher with R training alone than with RA training. Microsc. Res. Tech. 77:727–734, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION It is well known that both, A training alone, and R training alone improve the performance and increase the reserve capacity of the myocardium (Anversa et al., 1983; Camargo et al., 2008; Difee et al., 2001; Kemi et al., 2002; Kwak et al., 2006; Rosa et al., 2005; Thomas et al., 2000; Vaz da Silva et al., 2002; Vitartaite et al., 2004). R training is associated in humans and animals with a concentric form of non-pathologic LV hypertrophy characterized by augmented ventricular wall thickness without changes in cavity size (Barauna et al., 2007; Haykowsky et al., 2002; Pluim et al., 2000). An increase in cardiomyocyte cross-sectional area is observed at the microscopic level (Barauna et al., 2007; Grossman et al., 1975). The potential effects of R exercise on changes in myocardial collagen tissue are unknown. In contrast, A training leads to an eccentric form of non pathologic cardiac hypertrophy, which is primarily C V
2014 WILEY PERIODICALS, INC.
characterized by an increased left ventricular (LV) cavity dimension (and thus, LV mass) (Jin et al., 2000; Kemi et al., 2002; Morganroth et al., 1975), cardiomyocyte hypertrophy (Fenning et al., 2003; Medeiros et al., 2004; Mokelke et al., 1997; Moore et al., 1993, 1995; Palmer et al., 1998; Wislïff et al., 2002), increased arteriolar number and length (Brown and Hudlicka, 1999; Laughlin and Mcallister, 1992; Pinheiro et al., 2006; Tomanek, 1994), and decreased interstitial collagen tissue (Pinheiro et al., 2006). Combined A and R training, also known as concurrent training, has been used with positive outcomes for *Correspondence to: Romeu Rodrigues de Souza, Rua Afonso de Freitas, 451, Ap 122, S~ ao Paulo, SP, Brazil, 04006-052. E-mail: [email protected] Received 3 April 2014; accepted in revised form 8 June 2014 REVIEW EDITOR: Prof. George perry Contract grant sponsors: Fapesp, CNPq, and CAPES. DOI 10.1002/jemt.22394 Published online 20 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
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glycemic levels in diabetic patients (Cuff et al., 2003; Larose et al., 2010; Loimaala et al., 2009; Maiorana et al., 2000; Reid et al., 2010; Sigal et al., 2007; Tan et al., 2012) and to improve functional capacity and strength in patients with chronic heart failure (Maiorana et al., 2000). However, to the best of our knowledge, studies on the adaptation LV changes of combined RA training using an animal model has not been performed. We designed this study to determine the effects of aerobic and resistance training alone versus a sedentary control group, and the incremental effects of doing both types of exercise (combined exercise training) versus aerobic or resistance training alone, on LV cardiomyocyte area, density of capillaries, and interstitial collagen.
peritoneally) and then euthanized. The animals were heparinized prior to tissue fixation to optimize perfusion-fixation. The hearts were excised in diastole, and the myocardium was perfused through the aorta at a constant pressure of 80 mm Hg using 0.1 M cacodylate buffer (3 min) followed by 2.5% glutaraldehyde solution diluted in cacodylate buffer. Next, the LV, including the septum, was isolated and weighed. A perpendicular section to the long axis of the LV including the entire thickness of the LV wall was cut at the level of the papillary muscles. Microscopic determinations of the thickness of the LV wall and the LV internal diameter were made by measuring the widths of four uniformly spaced sites along the length of each section using a computerized program (Axio Vision, Carl Zeiss AG, Jena, Germany).
MATERIALS AND METHODS Exercise Training Twenty-eight 8-week-old male Wistar rats were housed in a temperature- and light-controlled room and fed ad libitum. They were randomly divided into four groups of seven, as follows: control (C), A training, R training, and RA training. The rats in the R and RA groups trained to climb a 1.1 m vertical (80 incline) ladder with weights tied to their tails (Hornberger and Farrar, 2004). They were trained once each day over 8 weeks and each training session consisted of six climbs. The weight that was carried by these rats during each session was progressively increased at the beginning of each week. Over the course of 8 weeks, the amount of weight carried by each rat was equivalent to 50% of its body weight (BW). The BW was measured at the end of each week and the new weight to be carried by the animal during the next week was adjusted according to its body weight (Heyward, 1998). After each R training session, the animals from the RA group were subjected to a moderate treadmill running program. They and the animals from the A group were trained to run 1 h/day for 5 days/week at 60% of the maximum effort test (MET) on the treadmill at 15% gradient. The MET was performed at baseline on a treadmill at a speed of 0.3 K/h. Every 4 min the belt speed was increased in the same proportion (0.3 km/h) (Fontinele et al., 2013; Silva et al., 1997). The MET was performed every 4 weeks by the C, A, and RA groups. After the MET, the A and RA animals were subjected to 8 weeks of treadmill training 5 days a week, with increasing velocity up to 60% of that achieved in the effort test. In the first week after the test, the animals ran for 30 min, increasing this time by 10 min each week until they reach 60 min in the fourth week, where at the end of it, another MET was performed to adjust the intensity of the training for the next 4 weeks. Rat in the C group was placed on the stationary treadmill daily for 10 min. Animal handling was approved by our University Ethics Committee in adherence to the International Guiding Principles for Biomedical Research Involving Animals.
Morphological and Quantitative Analysis The left ventricle was divided into slices that were approximately 3 mm wide and 5 mm long, and the slices were post-fixed in osmium tetroxide in sodium cacodylate buffer for 1 h. The tissue was dehydrated in graded alcohol solutions, embedded in Epon resin, and sectioned so that the cardiomyocytes of one half of the blocks were cut in cross-sections, while the cardiomyocytes of the other half were cut in longitudinal sections. Thin sections for transmission electron microscopy were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). The longitudinal sections were used to evaluate the fixation state of the myocardium by measuring the sarcomere length in the four groups. Two randomly chosen blocks from each ventricle in which the cardiomyocytes were cut in cross-section were used for the quantitative analysis. This process was researcher blinded. The ultra-thin sections were placed on a copper grid, and 10 randomly chosen fields per block (20 fields per LV) were selected for micrographs taken on a Jeol transmission electron microscope (Jeol, Tokyo, Japan). Each of the 280 electron micrographs was analyzed by the Axio Vision software program (Carl Zeiss, Jena, Germany) to determine the cardiomyocyte cross sectional area, the capillary volume density, and the collagen fibril diameter. The cardiomyocyte cross sectional area was determined in ten electron micrographs from each heart with final magnifications of 6003, obtained from regions where the cardiomyocytes were transversely sectioned. The capillary volume density was expressed as the fraction (%) of the total area (volume reference) occupied by capillaries. It was evaluated using a test system with 84 sampling points, which was displayed on a monitor and calibrated. The following formula was used: Vv[cap] 5 Pp[cap]/PT, where Pp[cap] is the number of points in contact with capillaries and PT is the total number of test-points (84) (Br€ uel et al., 2002; Wulfsohn et al., 2004). The volume density can be expressed in the range of 0–1, but it can also be expressed as a percentage (Ribeiro, 2006), taking 100% as the total number of test points (84) and calculating the percentage of capillaries viewed from the number of points in contact with capillaries. The collagen content was determined by light microscopy in samples of myocardial tissue dissected
Cardiac Hypertrophy At the end of the experiment, one day after the last training session, each animal was anesthetized with sodium pentobarbital (3 mg/100 g body weight, intra-
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The means were calculated and the data were tested for significance. Statistical Analysis All data were expressed as mean 6 standard deviation (SD). The data were tested for normal distribution using the Kolmogorov–Smirnov test. Differences among groups were assessed by one-way analysis of variance (ANOVA). When a significant difference was detected, comparisons were performed by Tukey’s post hoc test. P values less than 0.05 were considered statistically significant.
Fig. 1. Absolute weight lifted by rats in the C, A, R, and RA groups at the beginning, (week 0) and after 8 weeks of training *P < 0.05 compared with C, A, R, and RA at week 0 and with C, A, and RA at week 8. **P < 0.05 compared with C, A, R, and RA at week 0 and with C and A at week 8. One-way ANOVA followed by Tukey’s test was used for the statistical analysis.
TABLE 1. BW, LVW, LV wall thickness and LV internal diameter of the four studied groups of rats Parameter / Groups
c (n57)
A (n57)
R (n57)
RA (n57)
Body wt, g 314 6 11.2 296 6 10.6 306 6 24 325 6 37 LVW:BW mg/g 2.8 6 0.2 3.12 6 0.2a 3.8 6 0.3b 3.3 6 0.2c LV wall 2.43 6 0.04 2.53 6 0.12 2.84 6 0.06b 2.63 6 0.02c thickness, mm LV internal 6.4 6 0.06 7.6 6 0.08d 6.6 6 0.04 6.9 60.1c diameter, mm Values are the mean 6 SD. a P < 0.05 compared with C. b P < 0.01 compared with group C, and P < 0.05, compared with group A and RA. c P < 0.05 compared with C and A. d P < 0.05 compared with C and R (P < 0.05).
from the LV anterior wall that were fixed in 10% Bouin’s solution for 24 h, embedded in paraffin, and used to make tangential histological sections of 5 mm. The sections were stained using the Picrosirius technique (Junqueira et al., 1979) and examined under polarized light. When studied with this method, tissues containing collagen fibers show intensely birefringent thick and thin fibers (Junqueira et al., 1978). To determine the collagen content range in the four groups, histological sections were entered into a KS400 digital analysing computer program (Zeiss, Germany), which quantified the area percentage (of 12,000 mm2) of the collagen fibers. Three sections per heart were analyzed using a microscope equipped with appropriate polarization lenses. In each section, five randomized fields were selected, and the area percentage of collagen fibers was quantified in each field. The collagen fibril diameter was determined in ten electron micrographs of each heart at a final magnification of 130,0003 obtained from regions where the fibrils were transversely sectioned. The minimum diameter of each fibril was measured using the image analyzer program. By commencing at one corner of each field and radiating outward in an arc, the diameters of the fibrils present in the field were determined. Microscopy Research and Technique
RESULTS One Repetition Maximum Figure 1 shows the absolute weight that was lifted by the groups of rats during the repetition maximum test. The repetition maximum values for the C, A, R, and RA groups (810 6 10 g) were similar at the beginning (day 0) of the study. After 8 weeks, the load lifted by the animals in the C and A groups was similar to that lifted in the first test. The load lifted by the animals in the R group (1,110 6 100 g) was 27% higher than the load lifted in the first test and that lifted by the animals in the RA group was 12% higher than that the first test (in both cases, P < 0.05). In summary, after 8 weeks of training, the capacity to lifting load augmented significantly in R and RA groups but not in C and A groups. Body Weight and Cardiac Hypertrophy Results about the body weight and cardiac hypertrophy in C and trained animals are summarized in Table 1. No significant differences in body weight were observed among the four groups, at the end of the study (P > 0.05). The LVW: BW increased 11.4% in A group (P < 0.05), 35% in the R group (P < 0.01), and 18% in the RA group (P < 0.05) compared to the C group. The LV wall thickness increased 6% in the A group (n. s.) (P > 0.05), 17% in the R group (P < 0.05), and 10% in the RA group (P < 0.05) when compared to the C group. The LV internal diameter increased 19% in the A group (P < 0.05), 3% in the R group (n. s.) (P > 0.05), and 8% in the RA group (P < 0.05) compared with the C group. In summary, except for the LV internal diameter, the cardiac parameters were higher in the R group compared with the other groups. They were also higher in RA group compared with C and A groups. The LV internal diameter was higher in group A than in the other groups. Cross-sectional Area of Cardiomyocytes The cross-sectional area of cardiomyocyte increased 1% in the A group (n. s.) (P > 0.05), 27% in the R group (P < 0.01), and 12% in the RA group (P < 0.05), when compared to the C group (Figs. 2 and 3). In summary, after 8 weeks of exercise, the cross-sectional area of cardiomyocyte was higher in the R group than in C, A, and RA group and it was higher in RA group than in A and C groups. Myocardial Capillary Density Capillary density increased 5.4 6 0.6% in the A group (n. s.) (P > 0.05), 11 60.5% in the R group
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Fig. 4. Capillary density (%) in the control (C) AND trained (A, R, and RA) groups of rats. Values are the means 6 SD. *P < 0.05 compared with C, A, and RA. **P < 0.05 compared with C and A.
(P < 0.05), and 7.7 6 0.3% in the RA group (P < 0.05), compared with the C group (Fig. 4). In summary, capillary density increased more in R group than in RA group, and increased more in RA group than in group A, compared with C group.
Fig. 2. Low-power electron micrographs of transverse sections from the LV wall of the C, A, R, and RA groups of rats that were used to determine the cross-sectional area of cardiomyocytes (cm) and the capillary density. Capillaries are indicated by arrows. IT—Cardiac interstitium. Scale bar: 10 mm.
Fig. 3. Mean data showing the cardiomyocyte area (mM2) of rats from the groups C, A, R, and RA after 8 weeks exercise training. Values are the means 6 SD. *P < 0.05 compared with C and P < 0.01, compared with A and RA; **P < 0.05 compared with C and A.
Myocardial Collagen Content and Collagen Fibril Diameter Histological sections from the LV myocardium stained with Picrosirius showed that the amount of collagen fibers was higher in the R group compared with the C, A, and RA groups and that it seems to be higher in the RA group compared to the C, and A groups (Fig. 5). Analysis of electron micrographs of groups C and A with magnification of 130,0003 showed that the collagen fibers were composed primarily of small-diameter fibrils. In the hearts from the R and RA groups, both large-and small-diameter collagen fibrils were present but with a predominance of larger diameter fibrils (Fig. 6). Collagen density increased from 5.4 6 0.8% in the C group to 5.8 6 0.6% in the A group (n. s.) (P > 0.05), to 8.2 6 0.7% in the R group (P < 0.05), and to 6.3 6 0.4% in the RA group (P < 0.05). These results demonstrate a significant increase for collagen content in the LV with R and RA training, but the increase was higher with R training alone than with RA training (Figs. 5 and 7). Statistical analysis revealed that the collagen fibril diameter was the greatest in the hearts from the R group (81 6 8 nm) compared with those from the C group (54 6 7 nm), A group (58 6 5 nm), and RA group (63 6 6 nm) (in all cases, P < 0.05) and that it was greatest in the hearts from RA group compared with those from C and A group (P < 0.05) (Fig. 8). In summary, collagen fibril diameter increased more with R training followed by RA training. DISCUSSION The primary findings of this study were that in Wistar rat, A training, and combined RA training each improved LV parameters and that R training is superior to both types of training. The results show that values obtained for the LV weight, LV thickness and Microscopy Research and Technique
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Fig. 5. Picrosirius red-stained ventricular sections from C, A, R, and RA groups viewed under polarized light, that were used to show the LV interstitial collagen. Scale bar: 100 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
for the cardiomyocyte cross-sectional area, capillary density, and collagen density with R training were superior to that obtained with training A alone or RA training in comparison with the C group. The results obtained with RA training for the same parameters were superior to that of training A, compared with the C group. The values obtained for the LVW:BW and to LV internal diameter with training A were inferior to that obtained with training R and RA, in comparison with the C group. The increase in LV parameters by R training can be explained by the high intraventricular pressure that is needed to open the aortic valve. During the ejection phase, high intraventricular pressure leads to increase in myocardial wall stress, which is the major stimulus for cardiac hypertrophy in the pressure-overloaded heart (Kasikcioglu, 2004). Microscopy Research and Technique
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Fig. 6. High-power electron micrographs of transverse-sectioned collagen fibers from the LV wall of the C, A, R, and RA groups of rats. The electron micrographs are viewed with the same magnification showing collagen fibrils of large (*) and short diameter (arrows). Scale bar: 100 nm.
Conversely, during A training, in addition to the increased cardiac output, the blood pressure increases, although it does not increase to the same extent as during R training. Consequently, the aerobically trained LV must adapt to both volume and pressure loads (Mihl et al., 2008). The heart responds by increasing the LV internal diameter (Fenning et al., 2003; Mokelke et al., 1997; Moore et al., 1993, 1995; Moore and Palmer, 1999; Palmer et al., 1998; Pluim et al., 2000). Consequently, during A training, the volume load is the prevailing factor acting on the ventricular wall; therefore, the aerobic-trained heart develops eccentric hypertrophy (Vinereanu et al., 2002). The reason why RA induced a specific form of cardiac hypertrophy is not known. It seems that in this
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Fig. 7. Mean data for the collagen density (%) in the LV from C, A, RA, and R groups of rats. Values are the means 6 SD. *P < 0.05 compared with C, A, and RA. **P < 0.05 compared with C and A.
Fig. 8. Mean data for collagen fibril (CF) diameter in the LV from C, A, R, and RA groups of rats. Values are the mean 6 SD. *P < 0.05 compared with C, A, and RA. **P < 0.05 compared with C and A.
form of training the cardiac hypertrophy produced by R training was affected by training A. Can A training inhibit protein synthesis? A possibility is that A training limits signaling for muscle growth. However, further studies need to be done to resolve this issue. This study demonstrated that animals receiving each type of exercise training showed different influence in the cross-sectional area of cardiomyocyte. The increasing in cardiomyocyte area in response to R training, results from a pressure overload (increased post-load) on the heart (Fleck, 1988; MacDougall et al., 1992). This pressure overload leads to a higher final systolic pressure to create a parallel response in the sarcomeres and to augmented LV wall thickness that characterizes the concentric hypertrophy (Effron, 1989; Shapiro, 1984). The expansion of cross-sectional area of cardiomyocyte is caused by a parallel sarcomere replication, which can contribute to the enhancement of the intrinsic contractility of these cells (Perrault and Turcotte, 1994). In addition, the intrinsic contractility of the cardiomyocyte may be a potential mechanism underlying the training-induced improvement of myocardial contractile function. Therefore, the
architecture of heart that is subjected to volumetric overload is altered via hypertrophy, promoting LV remodeling to normalize the parietal tension in line with Laplace’s Law (Lorell and Carabello, 2000; Zhang et al., 2011). Conversely, the A training increases the venous return and, consequently, imposes a volumetric overload (increased pre-load) on the heart. This overload results in the elevation of the final diastolic pressure experienced by the heart to produce a response in the sarcomeres, increasing the length of the cardiomyocyte and the size of the LV cavity, which characterizes eccentric hypertrophy (Mokelke et al., 1997; Moore et al., 1993; Palmer et al., 1998, 1999). In this study, the myocardial capillary volume density was altered by both R and RA training. Notably, the capillary volume density was most increased following R training alone. The capillary volume density in the RA and R groups may have increased due to the neo-formation mechanism (angiogenesis) in agreement with previous results following A training (Amaral et al., 2000). The neo-formation of vessels occurs in animals with impaired myocardium and in healthy animals to improve the blood supply to the myocardium (Brown and Hudlicka, 1999; Laughlin and Mcallister, 1992; Tomanek, 1994). This study demonstrated that the interstitial collagen content and composition were influenced differently by each type of training indicating that the myocardial collagen is likely remodeled differently by different types of training. One possible explanation is that the myocardial collagen degradation is related to cardiac pressure overload. In fact, the regulation of fibril collagen mRNA levels following pressure overload may be a result of hemodynamic changes and their impact on cardiac fibroblasts (Eghbali, 1990). Transforming growth factor-b1 (TGF-b1), which is present in cardiac fibroblasts, stimulates collagen gene expression in the extracellular matrix (Eghbali et al., 1989). Therefore, TGF-b1 expression may increase with pressure overload, which could explain the increase in collagen transcription in the R and RA exercised heart. However, additional studies are needed to determine the mechanisms by which the training affects myocardial collagen. Our trial was not designed to study effects of exercise volume or duration per se, and the superior effect of R training in some cardiac parameters may reflect the greater effect of training performed by the R group. Furthermore, because the physiologic effects of RA training differ from those of the A training alone, we cannot assume that our results reflect only additional training time. If our findings simply reflected duration of active training, we would expect that the effect of A training and of R training alone on myocardial parameters would be less than half that of RA training (Sigal et al., 2007). Instead, the effects of the A training and RA training on the myocardium were quite similar, and those of R training were twice those of A training. CONCLUSION This data suggest that RA training was more effective than training A, in eliciting improvements on the LVW: BW, LV wall thickness, and cross-sectional area of cardiomyocyte, capillary density, and collagen fibril Microscopy Research and Technique
COMBINED TRAINING AND RAT MYOCARDIUM
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