Swimming Training Improves Myocardial Mechanics, Prevents Fibrosis, and Alters Expression of Ca2+ Handling Proteins in Older Rats

Swimming Training Improves Myocardial Mechanics, Prevents Fibrosis, and Alters Expression of Ca2+... Abstract Exercise training effects on the contractility of aged myocardium have been investigated for more than 20 years, but the data are still unclear. This study evaluated the hypothesis that a swimming training (ST) may improve myocardial inotropism in older rats. Male Wistar rats aged 4 (young)-and 21 (old)-months-old were divided into young untrained (YNT), old untrained (ONT), and old trained (OTR; 6 weeks of ST) groups. Echocardiography and hemodynamic were employed to assess left ventricular morphology and function. Myocardial mechanics was evaluated on papillary muscles. Histological and immunoblotting were carried out to evaluate fibrosis and proteins that modulate the myocardial function and calcium handling. We found that older rats did not show cardiac dysfunction, but ONT group showed lower physical performance during a swimming test (YNT: 5 ± 2; ONT: −16 ± 0.4; OTR: 51 ± 3; Δ%, sec). Moreover, ONT group showed worse myocardial inotropism, in which it was reversed by ST (Peak developed tension: YNT: 6.2 ± 0.7; ONT: 3.9 ± 0.3; OTR: 6.9 ± 0.9; g/mm2). The ST was associated with preserved collagen content (YNT: 0.38 ± 0.05; ONT: 0.78 ± 0.12; OTR: 0.34 ± 0.09; %). Exercise partially mitigated the effects of aging on intracellular Ca2+-regulating protein (eg, L-Ca2+ channel and phospholamban) and β-isoform of myosin. Thus, we propose that these molecular alterations together with inhibition of collagen increase contribute to improved myocardial performance in older rats. Aging, Cardiac remodeling, Contractile proteins, Myocardial performance, Physical training Exercise training is widely recommended for the elderly because it improves their functional fitness, exercise tolerance, and quality of life (1). Moreover, a significant improvement in exercise-induced cardiac function can be achieved. For example, sedentary seniors (aged ~70 years) who completed 1 year of endurance training display significant improvement in left ventricular (LV) systolic function (2). It has been proposed that changes in heart function may play a role in exercise training to improve myocardial contractile performance. Thus, previous investigations have analyzed effects of chronic exercise on isolated myocardial contractile performance. This approach shows to be suitable for determining myocardial inotropism with the control of loading conditions, for example, pre- and after-loading. Although the effects of exercise training on the contractile function of aged myocardial tissue have been investigated for more than 20 years, the available data are still uncertain. In this regard, Tate and colleagues (3) and Gwathmey and colleagues (4) have analyzed isometric contractile properties of papillary muscles of approximately 24-months-old Fischer 344 rats and showed that 8–10 weeks of running improved myocardial performance (as indicated by time-to-peak muscle tension) in older rats. However, this is not a uniform finding, in which other studies have shown no effect of exercise training on myocardial function decline with aging. Wei and colleagues (5) evaluated repercussion of treadmill running on myocardial mechanics of old (24- to 26-months-old) male Fischer rats and no repercussion on developed tension and rate of tension development was reported. Similar results were reported by other investigators (6), where old (23- to 24-months-old) rats that were treadmill trained for 4–8 weeks did not show a significant improvement in myocardial performance. Inconsistent exercise training effects on myocardial performance of older rats led the current study to evaluate whether swimming training may improve myocardial inotropism in older rats. In addition, the previous information has reported only the influence of running training on aging-related changes in myocardial mechanics in old rodents and the role of swimming training is still uncertain. To explore potential mechanisms underlying improvement of myocardial contractility, we have investigated the expression of intracellular Ca2+-regulation as well as sarcomere function proteins in the heart of sedentary and trained old rats. Our data suggest that Ca2+ transport and myosin heavy chain may be two of the potential mechanisms underlying improvement of myocardial performance with exercise training (3,7). Method Animals Male Wistar rats aged 4- and 24-months-old (young and old, respectively) were purchased from the Central Institute on Experimental Animal colony (CEDEME) maintained by Federal University of São Paulo. Animals were housed individually in standard cages with ad libitum access to rat chow and water in a humidity-controlled room with a 12:12 hour light:dark cycle. The 21-months-old group is represented as old-aged because of the median lifespan of ad libitum-fed male Wistar rats in CEDEME. A total of 29 animals were divided into young untrained (YNT; n = 10), old untrained (ONT; n = 9), and old trained (OTR; n = 10) groups. The study complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996), and the experimental protocol was approved by the Institutional Research Ethics Committee from the Federal University of São Paulo, São Paulo, Brazil (process: 1673020414). Exercise Training Protocol The OTR group was submitted to a swimming training approach in a container (132 cm in diameter and 80 cm deep) filled with tap water warmed to 32°C–34°C by a feedback-controlled electric heating coil (8). The water was maintained in turbulence to provide continuous swimming behavior and not float. Prior aim exercise protocol, rats were preconditioned to the swimming pool for six consecutive days, in which the session of exercise was limited to 15 minutes on the first day and increased by 15 minutes each day. Ultimately, rats swam 6 days a week, once a day, 90 min/day for 6 weeks. The rats swam without the addition of weight to the body or tail; this means that swimming rats were performing within at a moderate intensity [~45%–65% of VO2max (9)]. Physical Fitness Assessment The cardiorespiratory fitness was evaluated using a motorized treadmill coupled to a gas analyzer (Panlab, Harvard Bioscience Company, MA). All animals were initially familiarized with a motor-driven treadmill as previously described by our group (10). The physical fitness was also assessed using an exhaustive swim test (11). The load corresponding to 10% body weight was attached around the waist of each animal, in which animals were individually observed to determine swimming time until exhaustion. Exhaustion was defined as the point when the animal could not swim up to the water surface for 10 seconds. Biometric and Histomorphometric Data Animals were anesthetized with urethane overdose (4.8 g/kg i.p.), and the hearts were rapidly removed and weighed. Then, the LV was separated from the rest of the heart, weighed, and transversally sectioned at the mid-ventricular level. The basal portion was fixed in 10% formalin buffered solution for optical microscopic examination. Myocardial tissue was sectioned at 7 μm thickness and stained with hematoxylin–eosin and picrosirius red. Cardiomyocyte nuclear volume and collagen content were assessed at 40× magnification using an Olympus image acquisition system [Waltham, MA (12,13)]. Echocardiography Animals were anesthetized (i.p.) with ketamine (50 mg/kg) plus xylazine (10 mg/kg) 24 h after the last exercise session, and measurements were performed using a 12 MHz transducer connected to an HP Sonos-5500 echocardiograph [Hewlett-Packard, CA (14)]. The LV diastolic (DA) and systolic (SA) transverse areas were assessed by two-dimensional images in the basal, middle, and apical parasternal transverse planes. The real value was the arithmetic mean of measurements of the three views. The systolic performance was examined by the fractional area change (FAC = DA − SA/DA, %). Pulsed Doppler at the LV side of the mitral valve provided the flow velocity curve to analyze the diastolic performance (E and A waves and E/A ratio). LV Hemodynamic Study Twenty-four hours after echocardiographic study, rats were anesthetized (urethane, 1.2 g kg−1 i.p.) and a 2-F gauge (length 140 cm) Millar catheter-tip micromanometer (model SPR-320, Millar Instruments, Houston, TX) was inserted through the right carotid artery into the LV cavity (15). The follow parameters were recorded: heart rate (HR); systolic pressure (LVSP); end-diastolic pressure (LVEDP); maximum positive (+dP/dt), and negative (–dP/dt) time derivatives of the developed pressure. Data were evaluated with AcqKnowledge 3.5.7 software (Biopac Systems, Inc., CA). Myocardial Mechanics Immediately after hemodynamic study, animals received urethane overdose (4.8 g/kg i.p.), and the hearts were quickly removed. Anterior papillary muscles of the LV were isolated and prepared as previously described (8,15). The muscle was attached to an isometric strength transducer (Grass FT-03, Astro-Med, Inc., RI) and stretched to contract isometrically at peak length of its length–tension curve (Lmax). Then, peak of developed tension (DT) and maximal rate of tension increase (+dT/dt) and decrease (−dT/dt) were obtained. Moreover, length–DT curves were derived from data obtained at lengths corresponding to 92%, 94%, 965, 98%, and 100% of the Lmax. Relative PPC (post-pause contraction) was evaluated using pause durations of 10, 20, and 60 seconds. Relative PPC was expressed as the amplitude of post-pause DT divided by the steady-state DT. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Immunoblotting The frozen apical LV portion was homogenized as described in detail elsewhere (13,15), and 30 µg of the homogenate was subjected to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membrane was soaked in a blocking buffer (5% nonfat dry milk, 10 mM Tris–HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies (Abcam, Cambridge, MA): rabbit anti-sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a; 1:1,000), rabbit anti-Na+/Ca2+ exchanger (NCX; 1:1,000), L-type calcium channel (L-Ca2+; 1:1,000), ryanodine receptor (RyR; 1:1,000), phospholamban (PLB), phospho-Thr17-PLB (PLB-Thr17), phospho-Ser16-PLB (PLB-Ser16; 1:1,000), and anti-myosin heavy chain alpha (α-MHC; 1:5,000) and beta (β-MHC; 1:5,000). Membranes were washed five times with PBS and incubated for 1 hour with horseradish peroxidase-conjugated (1:2,000; Jackson ImmunoResearch Laboratories). Bound antibody was detected using an enhanced chemiluminescence reagent (GE HealthCare, Piscataway, NJ) for 1 minute, and bands were visualized and digitalized using the Amersham Imager 600 system. Quantification of target proteins was normalized for the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Abcam, Cambridge, MA). Statistical Analysis Data were statistically analyzed using GraphPad Prism 5.0 (La Jolla, CA). Data normality was examined with Shapiro–Wilk test. One-way ANOVA (Newman–Keuls post hoc) was applied to detect differences between groups. Kruskal–Wallis followed by Dunn’s multiple comparison tests was applied to non-normally distributed data. Statistical significance was set at p-value of ≤.05. Data are shown as mean ± standard error of the mean (SEM). Results Swimming Training Reverses Physical Fitness Deficit in Older Animals Figure 1A shows VO2peak analysis of treadmill running. It showed that VO2peak was not significantly affected by exercise training in older animals during the follow-up study segment. On the other hand, there was a significant reduction in swimming performance in the ONT group (Figure 1B). Notably, exercise training resulted in marked improvement in swimming performance in the OTR group. Figure 1. View largeDownload slide Physical fitness of the YNT (n = 10), ONT (n = 9), and OTR (n = 10) groups as a result of 6 weeks of swimming training or similar period for nonexercised rats. (A) Peak oxygen uptake (VO2peak). (B) Swimming test. Data are showed as mean ± SEM. Two-wall ANOVA followed by Bonferroni multiple comparison tests were applied to detect differences between the groups. YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group to respective time; #p < .05 vs ONT group. Figure 1. View largeDownload slide Physical fitness of the YNT (n = 10), ONT (n = 9), and OTR (n = 10) groups as a result of 6 weeks of swimming training or similar period for nonexercised rats. (A) Peak oxygen uptake (VO2peak). (B) Swimming test. Data are showed as mean ± SEM. Two-wall ANOVA followed by Bonferroni multiple comparison tests were applied to detect differences between the groups. YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group to respective time; #p < .05 vs ONT group. Swimming Training Induces Myocardial Hypertrophy and Prevents Fibrosis Rats in the ONT group exhibited a higher ratio of LV weight to body weight compared with rats in the YNT group. Trained animals also showed an additional effect on the heart weight to body weight ratio (Figure 2A and B). Exercise-induced myocardial hypertrophy was confirmed by nuclear cardiomyocyte volume analyses (Figure 2C). Concerning collagen content, older rats showed higher fibrosis depositions in the LV chamber (Figure 2D). The beneficial role of exercise training was observed with no significant differences in myocardial collagen between the YNT and OTR groups. Figure 2. View largeDownload slide Effects of swimming training on cardiac mass (YNT, n = 10; ONT, n = 9; OTR, n = 10) and fibrosis (n = 5 per group) induced by aging. (A) HW/BW heart weight/body weight. (B) LV/BW, left ventricular weight/body weight. Representative photomicrographs showing (C) nuclear volume and (D) collagen staining in the LV myocardial tissue, respectively (magnification ×40; scale bar: 250 μm). One-way ANOVA and Newman–Keuls post hoc were applied to detect differences between the groups. Significance level for the comparisons is shown above the bars. HW/BW = heart weight/body weight; LV/BW = left ventricular weight/body weight; YNT = young untrained; ONT = old untrained; OTR = old trained. Figure 2. View largeDownload slide Effects of swimming training on cardiac mass (YNT, n = 10; ONT, n = 9; OTR, n = 10) and fibrosis (n = 5 per group) induced by aging. (A) HW/BW heart weight/body weight. (B) LV/BW, left ventricular weight/body weight. Representative photomicrographs showing (C) nuclear volume and (D) collagen staining in the LV myocardial tissue, respectively (magnification ×40; scale bar: 250 μm). One-way ANOVA and Newman–Keuls post hoc were applied to detect differences between the groups. Significance level for the comparisons is shown above the bars. HW/BW = heart weight/body weight; LV/BW = left ventricular weight/body weight; YNT = young untrained; ONT = old untrained; OTR = old trained. Swimming Training Effects on LV Morphology and Performance Body weight was similar among the groups at the end of the study. Older rats showed suitable LV morphology alterations only on transthoracic echocardiography (Table 1). In this regard, an increased LV systolic area was the only modification compared to young animals, in which it was not observed upon training. Aging and exercise training had no impact on the LV performance on echocardiographic analysis. Hemodynamic measurements (Table 1) showed that LV end-diastolic pressure was significantly higher in older rats compared with the YNT group. Exercise training did not affect LV end-diastolic pressure. Table 1. Influence of Aging and Exercise Training on LV Morphology and Function   YNT (n = 10)  ONT (n = 9)  OTR (n = 10)  Body weight (g)  391 ± 13  415 ± 27  427 ± 22  Echocardiography   HR (bpm/min)  255 ± 12  247 ± 19  240 ± 20   LVDA (cm2)  0.49 ± 0.002  0.55 ± 0.02  0.51 ± 0.02   LVDA/BW (cm2/g)  0.0012 ± 0.0006  0.0013 ± 0.0006  0.0012 ± 0.0009   LVSA (cm2)  0.12 ± 0.01  0.18 ± 0.01*  0.15 ± 0.01   LVSA/BW (cm2/g)  0.00030 ± 0.00003  0.00042 ± 0.000027*  0.00035 ± 0.000034   FAC (%)  75 ± 1  68 ± 2  70 ± 3   E wave (cm/s)  0.64 ± 0.04  0.53 ± 0.03  0.62 ± 0.03   A wave (cm/s)  0.35 ± 0.02  0.31 ± 0.03  0.35 ± 0.01   E/A ratio  1.84 ± 0.12  1.8 ± 0.17  1.81 ± 0.13  Hemodynamic   HR (bpm/min)  349 ± 17  389 ± 4  366 ± 11   LVSP (mmHg)  119 ± 3  123 ± 6  127 ± 5   LVEDP (mmHg)  2.4 ± 0.2  3.7 ± 0.2*  4.7 ± 0.4*   +dP/dt (mmHg/s)  8,101 ± 624  9,340 ± 680  8,175 ± 752   −dP/dt (mmHg/s)  6,603 ± 390  5,811 ± 574  5,926 ± 321    YNT (n = 10)  ONT (n = 9)  OTR (n = 10)  Body weight (g)  391 ± 13  415 ± 27  427 ± 22  Echocardiography   HR (bpm/min)  255 ± 12  247 ± 19  240 ± 20   LVDA (cm2)  0.49 ± 0.002  0.55 ± 0.02  0.51 ± 0.02   LVDA/BW (cm2/g)  0.0012 ± 0.0006  0.0013 ± 0.0006  0.0012 ± 0.0009   LVSA (cm2)  0.12 ± 0.01  0.18 ± 0.01*  0.15 ± 0.01   LVSA/BW (cm2/g)  0.00030 ± 0.00003  0.00042 ± 0.000027*  0.00035 ± 0.000034   FAC (%)  75 ± 1  68 ± 2  70 ± 3   E wave (cm/s)  0.64 ± 0.04  0.53 ± 0.03  0.62 ± 0.03   A wave (cm/s)  0.35 ± 0.02  0.31 ± 0.03  0.35 ± 0.01   E/A ratio  1.84 ± 0.12  1.8 ± 0.17  1.81 ± 0.13  Hemodynamic   HR (bpm/min)  349 ± 17  389 ± 4  366 ± 11   LVSP (mmHg)  119 ± 3  123 ± 6  127 ± 5   LVEDP (mmHg)  2.4 ± 0.2  3.7 ± 0.2*  4.7 ± 0.4*   +dP/dt (mmHg/s)  8,101 ± 624  9,340 ± 680  8,175 ± 752   −dP/dt (mmHg/s)  6,603 ± 390  5,811 ± 574  5,926 ± 321  Note: Echocardiographic and hemodynamic analysis of left ventricular (LV) at the end of the study (YNT, n = 6; ONT, n = 9; OTR, n = 9). YNT = young untrained; ONT = old untrained; OTR = old trained; LV = left ventricule; LVDA = LV diastolic area; LVSA = LV systolic area; FAC = fractional area change; E/A ratio = ratio between the E and A waves; HR = heart rate; LVSP = LV systolic pressure; LVEDP = LV end-diastolic pressure; +dP/dt = maximal positive time derivate of developed pressure; −dP/dt = maximal negative derivate of developed pressure; BW = body weight. Data were analyzed with one-way ANOVA and post hoc Newman–Keuls test. *Different from the YNT group. View Large Swimming Training Improvements the Myocardial Mechanics in Older Animals There were remarkable differences in myocardial performance between the three groups. We observed that older rats had myocardial muscle that developed less force than their respective young controls; the result is depicted as a smaller DT and +dT/dt (Figure 3A and B). Moreover, −dT/dt was also significantly lower in older animals (Figure 3C). The myocardial mechanics was significantly affected by exercise training. Indeed, papillary muscles from the OTR group exhibited similar DT and +dT/dt data compared with the YNT group, as well as an improved −dT/dt. Figure 3. View largeDownload slide In vitro myocardial performance of the papillary muscle evaluated at lengths corresponding to 100% of Lmax (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Typical tracings representing mechanical parameters of the papillary muscle. (B) Peak developed tension (DT). Maximal (C, +dT/dt) positive and (D, −dT/dt) negative time derivative of DT. (E) Straight lines were fitted to the developed length–tension relationships using linear regression analysis. Mean DT slopes were compared between groups with one-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars (right graph). (F) Potentiation of post-rest contractions is evident in young untrained (YNT) rats while post-rest steady-state of force was reported in muscles of older rats with no effect of exercise training. Two-way repeated ANOVA and Bonferroni post hoc test was applied to detect differences between the groups and times. Significance level for comparisons between pause is shown above the bars (right graph). YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group for the corresponding pause. Figure 3. View largeDownload slide In vitro myocardial performance of the papillary muscle evaluated at lengths corresponding to 100% of Lmax (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Typical tracings representing mechanical parameters of the papillary muscle. (B) Peak developed tension (DT). Maximal (C, +dT/dt) positive and (D, −dT/dt) negative time derivative of DT. (E) Straight lines were fitted to the developed length–tension relationships using linear regression analysis. Mean DT slopes were compared between groups with one-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars (right graph). (F) Potentiation of post-rest contractions is evident in young untrained (YNT) rats while post-rest steady-state of force was reported in muscles of older rats with no effect of exercise training. Two-way repeated ANOVA and Bonferroni post hoc test was applied to detect differences between the groups and times. Significance level for comparisons between pause is shown above the bars (right graph). YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group for the corresponding pause. As shown in Figure 3D, the DT was plotted as a dependent of muscle length. The active length–DT relationship from the ONT group was shifted downwards, with a lower slope value of linear worsened when compared to the other groups. Notably, the slope was preserved in exercise-trained rats. In this way, it is possible to state that for the same variation of muscle length, recruitment of the Frank–Starling mechanism is preserved in the older trained myocardium. In Figure 3E, there are post-rest contraction data that allow indirect evaluation of the SR function (16). In the healthy myocardium of rats, post-rest contraction is potentiated due to additional Ca2+ accumulated in the SR during the stimulus pause depending on SERCA2 activity and increased fractional Ca2+ release upon activation. Thus, the post-rest contraction was well evident in the YNT group, which increased with the duration of the pause. However, in the muscles from older groups, post-rest contraction behavior was changed. There was a plateau of the muscle force after the first stimulus pause, in which it was independent of the exercise training action. Swimming Training Modulates Expression of Ca2+-Regulating Proteins and Normalizes Myocardial Isoform Switches Changes in expression of proteins that modulate calcium handling have been a common finding in the older myocardium (17). In this study, older rats showed a significant decrease in NCX, SERCA2a, PLB-Thr17, and PLB-Ser16 expression (Figure 4). Furthermore, ONT animals exhibited higher L-Ca2+ content. Exercise training attenuated effects of aging on L-Ca2+, PLB-Thr17, and PLB-Ser16 expression. We also assessed whether older animals could have altered protein expression of α- and β-MHC (Figure 4I and J). These analyses were carried out because the distribution of α- and β-MHC showed to be directly correlated with myocardial dysfunction in aged tissue (18). There was no differential expression of α-MHC between the experimental groups (Figure 4I). However, Figure 4J illustrates that β-MHC expression was upregulated in the ONT group and unaltered in the OTR group. Therefore, β/α ratio was higher in the ONT group (Figure 4K) and preserved in trained animals. Figure 4. View largeDownload slide Myocardial protein expression evaluated by immunoblotting (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Representative blots for proteins that regulate the Ca2+ handling. The evaluated proteins were (B) Na+/Ca2+ exchanger (NCX), (C) L-type calcium channel (L-Ca2+), (D) ryanodine receptor (RyR), (E) sarcoplasmic reticulum Ca2+-ATPase (SERCA 2a), (F) phospholamban (PLB), (G) phospho-Thr17-PLB (PLB-Thr17), and (H) phospho-Ser16-PLB (PLB-Ser16). Protein expression of α-MHC and β-MHC isoforms is shown in I and J, respectively. (K) β-MHC/α-MHC ratio. All values were normalized for levels of GAPDH. One-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars. YNT = young untrained; ONT = old untrained; OTR = old trained. Figure 4. View largeDownload slide Myocardial protein expression evaluated by immunoblotting (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Representative blots for proteins that regulate the Ca2+ handling. The evaluated proteins were (B) Na+/Ca2+ exchanger (NCX), (C) L-type calcium channel (L-Ca2+), (D) ryanodine receptor (RyR), (E) sarcoplasmic reticulum Ca2+-ATPase (SERCA 2a), (F) phospholamban (PLB), (G) phospho-Thr17-PLB (PLB-Thr17), and (H) phospho-Ser16-PLB (PLB-Ser16). Protein expression of α-MHC and β-MHC isoforms is shown in I and J, respectively. (K) β-MHC/α-MHC ratio. All values were normalized for levels of GAPDH. One-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars. YNT = young untrained; ONT = old untrained; OTR = old trained. Discussion The current study has three main findings. First, swimming training-induced myocardial hypertrophy, and cardiomyocyte growth has been accompanied by a protective exercise effect on the myocardial structure. Thus, an increased interstitial collagen content was not present in trained older rats. Second, swimming training attenuated the effect of aging on several key proteins that modulate cardiomyocyte Ca2+ handling. A protective effect of exercise was most pronounced on β-MHC expression and β-MHC/α-MHC ratio, in which swimming training has mitigated increases evoked by aging. Third, older rats did not show substantial LV dysfunction but aging impaired the myocardial inotropism. Swimming training reversed the negative effect of aging, improving myocardial inotropism to a level similar to that of young rats. As illustrated in Figure 1, improved functional fitness was not identified when considering the VO2peak, which is a gold standard marker to assess exercise tolerance (19). On the other hand, when exercise performance was analyzed using a swimming approach, we identified a minor aerobic resistance in untrained older rats (~17%), while trained older rats showed a marked improvement (~50%). It is difficult to understand the disagreement between VO2peak and exhaustive swim test, but the training modality may have influenced the VO2peak measurement. Koch and colleagues (20) showed that maximal oxygen uptake increased more with aerobic interval training relative to continuous moderate exercise. In addition, the exercise training approach may have influenced our analysis, thereby, since the exercise training was carried out in the pool, the swimming test was more suitable to identify the improvement of physical fitness. Thus, a swimming test can be a useful tool to determine the physical fitness level of aging. Our results corroborate previous data that demonstrated myocardial hypertrophy in aged rats (21,22), which was more pronounced with exercise. Exercise training also had a significant effect on collagen concentration, where values in older trained rats were like those seen in young rats. Although other researchers showed similar findings, some data have been inconsistent. Thomas and colleagues (23) noted an increase in hydroxyproline LV concentration comparing 10 weeks with 22-months-old rats that was somewhat attenuated in the senescent rats who were submitted to a progressive treadmill incline (15%) at approximately 70% of maximal oxygen uptake (45 min/day; 5 days/week) for 10 weeks. However, Tomanek and colleagues (24) failed to find a significant collagen reduction in 22-months-old rats because of a motor-driven treadmill 6 days per week for a 12-week period. The no-singular findings may be due to species variances, maturational status, or even differences in exercise protocols. We have applied echocardiography and hemodynamic approaches to evaluate LV performance, and results from these experiments have revealed an only significant change in LVEDP, previous corroborating evidence that cardiac compliance decreases with aging (25,26). It is conceivable that the age-related decline in the compliance is likely a greater myocardial stiffness, and our findings of increased collagen LV concentration show to be a main cause of the altered stiffness in nontrained rats (23,25). However, LVEDP levels reached from 3.7 to 4.7 mmHg in elderly rats, which has been considered similar to the normal range for health young rats (27). Although LV performance has not been extensively altered in older rats, myocardial performance was worsened as assessed by both DT and +dT/dt on in vitro papillary muscle preparations. Moreover, aging evoked a downwards shift of resting length–DT curves, suggesting that the Frank–Starling relationship is also worsened (15). Lastly, aging may change the regular post-rest potentiation to post-rest stabilization of force. In fact, as most of the Ca2+ returns to the SR during rest, its content tends to accumulate with prolonged pause time, also increasing Ca2+ release and force generation in next activation (16). Hence, post-rest potentiation alterations may be attributed to malfunctioning SR, in which it converges in a relative reduction of free Ca2+. Our experiments suggest that impaired SR reuptake function may be due to decreased SERCA2a content and its regulatory proteins (PLB-Thr17/Ser16). Moreover, altered SERCA2a and PLB-Thr17/Ser16 also helps explain slowed myocardial relaxation (−dT/dt) in older rats. An interesting aspect of this study is the reduced NCX and increased L-Ca2+ myocardial content, which to our knowledge is a finding described only by few investigations (28). The repercussion of these discoveries of muscle function shown to be clarified, but a first step could be to investigate the impact of altered NCX and L-Ca2+ on Ca2+ handling because a prolonged Ca2+ transients (3) or slower Ca2+ transient decay arises (17) are commonly reported in older rats. Finally, our findings corroborate that age-related change in contractile proteins is an upright shift of slower β-MHC, which can slow relaxation (17). Moreover, increased β-MHC expression may also partially explain lower +dT/dt of aged papillary muscles. Studies carried out on myofibrillar ATPase activity have reported that the speed with which muscle shortens is correlated with the ATP-hydrolyzing capacity of the myosin (18). The most important finding of the present study was the effectiveness of swimming training on improvement of myocardial contractility in older rats. Exercise resulted in maintenance of myocardial inotropism and contraction speed as well as improvement of relaxation. Moreover, Frank–Starling mechanism was also preserved in trained aged rats. To our knowledge, previous studies have examined the role of running exercise training in myocardial performance, in which the duration of training has varied between 4 and 20 weeks on a rodent treadmill. These studies have identified benefits (3,4) or not (5,6) of exercise training on contractile indices of myocardial performance (eg, longer time-to-peak tension). In our study, an increased rate of contraction (+dT/dt) and relaxation (−dT/dt) myocardial was accompanied by improved inotropism (DT) in older rats undergoing 6 weeks swimming training. These results suggest that better results for myocardial mechanics can be obtained with a swimming training program compared to running in rats. The underlying mechanism for the improvement in contractility is said to be multifactorial, including increased myocardial catecholamine stores (4), improvement in metabolic enzyme activity (29) as well as mitochondrial function (30), maintenance of myocyte number because of the anti-apoptotic exercise effect (31), and increase in atrial myosin light chain 1 expression (32). In our study, we have identified other mechanisms that may be involved in the improvement of exercise-induced myocardial performance. Inhibition of age-related changes in collagen content could help to explain the exercise-induced improvement of myocardial relaxation. We also provided evidence that intrinsic cardioprotective benefits in older rats can be associated with changes in several proteins that modulate Ca2+ handling. Maintenance of PLB-Ser16 levels and improvement of the PLB-Thr17 content may be associated with functional SERCA2a improvement. These findings may explain an increased calcium transport by the SERCA, in which it shows potential mechanisms underlying the improvement of myocardial performance with exercise training initiated during senescence (3). Notwithstanding, the preserved β-MHC/α-MHC ratio could be involved in the maintenance of myocardial shortening and relaxation speed as well as conserved Frank–Starling mechanisms in trained older rats. In summary, swimming training protected age-induced remodeling of myocardial tissue. It includes increased connective tissue and attenuated myocardial performance. Regular exercise also improved key proteins that regulate the Ca2+ handling and sarcomere velocity. It should be noted, however, that in the current design, it cannot be determined whether the benefits of exercise training are more evident to counteract aging over the longer term, in which exercise could be started in middle-aged animals to avoid adverse myocardial effects noticed in older animals. However, our study indicates that exercise is useful to improve myocardial mechanics when initiated in senescence. Although exercise resulted in improvement of myocardial inotropism associated with attenuation of the effect of aging on proteins that modulate intracellular calcium (LCa2+, PLB-Thr17, and PLB-Ser16), we were unable to directly test whether exercise training exercise modulates cardiomyocyte calcium handling in the older rats. Funding This work was supported by the National Council for Scientific and Technological Development (CNPq; 479395/2012–8) and the São Paulo Research Foundation (FAPESP; 15/11028–9 and 13/10619-8). Acknowledgments We thank Editage Translation Company for critical reviewing of English language. Conflict of Interest None declared. References 1. Floegel TA, Perez GA. An integrative review of physical activity/exercise intervention effects on function and health-related quality of life in older adults with heart failure. 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J Gerontol A Biol Sci Med Sci . 2012; 67: 1178– 1187. doi: 10.1093/gerona/gls146 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences Oxford University Press

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Abstract

Abstract Exercise training effects on the contractility of aged myocardium have been investigated for more than 20 years, but the data are still unclear. This study evaluated the hypothesis that a swimming training (ST) may improve myocardial inotropism in older rats. Male Wistar rats aged 4 (young)-and 21 (old)-months-old were divided into young untrained (YNT), old untrained (ONT), and old trained (OTR; 6 weeks of ST) groups. Echocardiography and hemodynamic were employed to assess left ventricular morphology and function. Myocardial mechanics was evaluated on papillary muscles. Histological and immunoblotting were carried out to evaluate fibrosis and proteins that modulate the myocardial function and calcium handling. We found that older rats did not show cardiac dysfunction, but ONT group showed lower physical performance during a swimming test (YNT: 5 ± 2; ONT: −16 ± 0.4; OTR: 51 ± 3; Δ%, sec). Moreover, ONT group showed worse myocardial inotropism, in which it was reversed by ST (Peak developed tension: YNT: 6.2 ± 0.7; ONT: 3.9 ± 0.3; OTR: 6.9 ± 0.9; g/mm2). The ST was associated with preserved collagen content (YNT: 0.38 ± 0.05; ONT: 0.78 ± 0.12; OTR: 0.34 ± 0.09; %). Exercise partially mitigated the effects of aging on intracellular Ca2+-regulating protein (eg, L-Ca2+ channel and phospholamban) and β-isoform of myosin. Thus, we propose that these molecular alterations together with inhibition of collagen increase contribute to improved myocardial performance in older rats. Aging, Cardiac remodeling, Contractile proteins, Myocardial performance, Physical training Exercise training is widely recommended for the elderly because it improves their functional fitness, exercise tolerance, and quality of life (1). Moreover, a significant improvement in exercise-induced cardiac function can be achieved. For example, sedentary seniors (aged ~70 years) who completed 1 year of endurance training display significant improvement in left ventricular (LV) systolic function (2). It has been proposed that changes in heart function may play a role in exercise training to improve myocardial contractile performance. Thus, previous investigations have analyzed effects of chronic exercise on isolated myocardial contractile performance. This approach shows to be suitable for determining myocardial inotropism with the control of loading conditions, for example, pre- and after-loading. Although the effects of exercise training on the contractile function of aged myocardial tissue have been investigated for more than 20 years, the available data are still uncertain. In this regard, Tate and colleagues (3) and Gwathmey and colleagues (4) have analyzed isometric contractile properties of papillary muscles of approximately 24-months-old Fischer 344 rats and showed that 8–10 weeks of running improved myocardial performance (as indicated by time-to-peak muscle tension) in older rats. However, this is not a uniform finding, in which other studies have shown no effect of exercise training on myocardial function decline with aging. Wei and colleagues (5) evaluated repercussion of treadmill running on myocardial mechanics of old (24- to 26-months-old) male Fischer rats and no repercussion on developed tension and rate of tension development was reported. Similar results were reported by other investigators (6), where old (23- to 24-months-old) rats that were treadmill trained for 4–8 weeks did not show a significant improvement in myocardial performance. Inconsistent exercise training effects on myocardial performance of older rats led the current study to evaluate whether swimming training may improve myocardial inotropism in older rats. In addition, the previous information has reported only the influence of running training on aging-related changes in myocardial mechanics in old rodents and the role of swimming training is still uncertain. To explore potential mechanisms underlying improvement of myocardial contractility, we have investigated the expression of intracellular Ca2+-regulation as well as sarcomere function proteins in the heart of sedentary and trained old rats. Our data suggest that Ca2+ transport and myosin heavy chain may be two of the potential mechanisms underlying improvement of myocardial performance with exercise training (3,7). Method Animals Male Wistar rats aged 4- and 24-months-old (young and old, respectively) were purchased from the Central Institute on Experimental Animal colony (CEDEME) maintained by Federal University of São Paulo. Animals were housed individually in standard cages with ad libitum access to rat chow and water in a humidity-controlled room with a 12:12 hour light:dark cycle. The 21-months-old group is represented as old-aged because of the median lifespan of ad libitum-fed male Wistar rats in CEDEME. A total of 29 animals were divided into young untrained (YNT; n = 10), old untrained (ONT; n = 9), and old trained (OTR; n = 10) groups. The study complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996), and the experimental protocol was approved by the Institutional Research Ethics Committee from the Federal University of São Paulo, São Paulo, Brazil (process: 1673020414). Exercise Training Protocol The OTR group was submitted to a swimming training approach in a container (132 cm in diameter and 80 cm deep) filled with tap water warmed to 32°C–34°C by a feedback-controlled electric heating coil (8). The water was maintained in turbulence to provide continuous swimming behavior and not float. Prior aim exercise protocol, rats were preconditioned to the swimming pool for six consecutive days, in which the session of exercise was limited to 15 minutes on the first day and increased by 15 minutes each day. Ultimately, rats swam 6 days a week, once a day, 90 min/day for 6 weeks. The rats swam without the addition of weight to the body or tail; this means that swimming rats were performing within at a moderate intensity [~45%–65% of VO2max (9)]. Physical Fitness Assessment The cardiorespiratory fitness was evaluated using a motorized treadmill coupled to a gas analyzer (Panlab, Harvard Bioscience Company, MA). All animals were initially familiarized with a motor-driven treadmill as previously described by our group (10). The physical fitness was also assessed using an exhaustive swim test (11). The load corresponding to 10% body weight was attached around the waist of each animal, in which animals were individually observed to determine swimming time until exhaustion. Exhaustion was defined as the point when the animal could not swim up to the water surface for 10 seconds. Biometric and Histomorphometric Data Animals were anesthetized with urethane overdose (4.8 g/kg i.p.), and the hearts were rapidly removed and weighed. Then, the LV was separated from the rest of the heart, weighed, and transversally sectioned at the mid-ventricular level. The basal portion was fixed in 10% formalin buffered solution for optical microscopic examination. Myocardial tissue was sectioned at 7 μm thickness and stained with hematoxylin–eosin and picrosirius red. Cardiomyocyte nuclear volume and collagen content were assessed at 40× magnification using an Olympus image acquisition system [Waltham, MA (12,13)]. Echocardiography Animals were anesthetized (i.p.) with ketamine (50 mg/kg) plus xylazine (10 mg/kg) 24 h after the last exercise session, and measurements were performed using a 12 MHz transducer connected to an HP Sonos-5500 echocardiograph [Hewlett-Packard, CA (14)]. The LV diastolic (DA) and systolic (SA) transverse areas were assessed by two-dimensional images in the basal, middle, and apical parasternal transverse planes. The real value was the arithmetic mean of measurements of the three views. The systolic performance was examined by the fractional area change (FAC = DA − SA/DA, %). Pulsed Doppler at the LV side of the mitral valve provided the flow velocity curve to analyze the diastolic performance (E and A waves and E/A ratio). LV Hemodynamic Study Twenty-four hours after echocardiographic study, rats were anesthetized (urethane, 1.2 g kg−1 i.p.) and a 2-F gauge (length 140 cm) Millar catheter-tip micromanometer (model SPR-320, Millar Instruments, Houston, TX) was inserted through the right carotid artery into the LV cavity (15). The follow parameters were recorded: heart rate (HR); systolic pressure (LVSP); end-diastolic pressure (LVEDP); maximum positive (+dP/dt), and negative (–dP/dt) time derivatives of the developed pressure. Data were evaluated with AcqKnowledge 3.5.7 software (Biopac Systems, Inc., CA). Myocardial Mechanics Immediately after hemodynamic study, animals received urethane overdose (4.8 g/kg i.p.), and the hearts were quickly removed. Anterior papillary muscles of the LV were isolated and prepared as previously described (8,15). The muscle was attached to an isometric strength transducer (Grass FT-03, Astro-Med, Inc., RI) and stretched to contract isometrically at peak length of its length–tension curve (Lmax). Then, peak of developed tension (DT) and maximal rate of tension increase (+dT/dt) and decrease (−dT/dt) were obtained. Moreover, length–DT curves were derived from data obtained at lengths corresponding to 92%, 94%, 965, 98%, and 100% of the Lmax. Relative PPC (post-pause contraction) was evaluated using pause durations of 10, 20, and 60 seconds. Relative PPC was expressed as the amplitude of post-pause DT divided by the steady-state DT. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Immunoblotting The frozen apical LV portion was homogenized as described in detail elsewhere (13,15), and 30 µg of the homogenate was subjected to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membrane was soaked in a blocking buffer (5% nonfat dry milk, 10 mM Tris–HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies (Abcam, Cambridge, MA): rabbit anti-sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a; 1:1,000), rabbit anti-Na+/Ca2+ exchanger (NCX; 1:1,000), L-type calcium channel (L-Ca2+; 1:1,000), ryanodine receptor (RyR; 1:1,000), phospholamban (PLB), phospho-Thr17-PLB (PLB-Thr17), phospho-Ser16-PLB (PLB-Ser16; 1:1,000), and anti-myosin heavy chain alpha (α-MHC; 1:5,000) and beta (β-MHC; 1:5,000). Membranes were washed five times with PBS and incubated for 1 hour with horseradish peroxidase-conjugated (1:2,000; Jackson ImmunoResearch Laboratories). Bound antibody was detected using an enhanced chemiluminescence reagent (GE HealthCare, Piscataway, NJ) for 1 minute, and bands were visualized and digitalized using the Amersham Imager 600 system. Quantification of target proteins was normalized for the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Abcam, Cambridge, MA). Statistical Analysis Data were statistically analyzed using GraphPad Prism 5.0 (La Jolla, CA). Data normality was examined with Shapiro–Wilk test. One-way ANOVA (Newman–Keuls post hoc) was applied to detect differences between groups. Kruskal–Wallis followed by Dunn’s multiple comparison tests was applied to non-normally distributed data. Statistical significance was set at p-value of ≤.05. Data are shown as mean ± standard error of the mean (SEM). Results Swimming Training Reverses Physical Fitness Deficit in Older Animals Figure 1A shows VO2peak analysis of treadmill running. It showed that VO2peak was not significantly affected by exercise training in older animals during the follow-up study segment. On the other hand, there was a significant reduction in swimming performance in the ONT group (Figure 1B). Notably, exercise training resulted in marked improvement in swimming performance in the OTR group. Figure 1. View largeDownload slide Physical fitness of the YNT (n = 10), ONT (n = 9), and OTR (n = 10) groups as a result of 6 weeks of swimming training or similar period for nonexercised rats. (A) Peak oxygen uptake (VO2peak). (B) Swimming test. Data are showed as mean ± SEM. Two-wall ANOVA followed by Bonferroni multiple comparison tests were applied to detect differences between the groups. YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group to respective time; #p < .05 vs ONT group. Figure 1. View largeDownload slide Physical fitness of the YNT (n = 10), ONT (n = 9), and OTR (n = 10) groups as a result of 6 weeks of swimming training or similar period for nonexercised rats. (A) Peak oxygen uptake (VO2peak). (B) Swimming test. Data are showed as mean ± SEM. Two-wall ANOVA followed by Bonferroni multiple comparison tests were applied to detect differences between the groups. YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group to respective time; #p < .05 vs ONT group. Swimming Training Induces Myocardial Hypertrophy and Prevents Fibrosis Rats in the ONT group exhibited a higher ratio of LV weight to body weight compared with rats in the YNT group. Trained animals also showed an additional effect on the heart weight to body weight ratio (Figure 2A and B). Exercise-induced myocardial hypertrophy was confirmed by nuclear cardiomyocyte volume analyses (Figure 2C). Concerning collagen content, older rats showed higher fibrosis depositions in the LV chamber (Figure 2D). The beneficial role of exercise training was observed with no significant differences in myocardial collagen between the YNT and OTR groups. Figure 2. View largeDownload slide Effects of swimming training on cardiac mass (YNT, n = 10; ONT, n = 9; OTR, n = 10) and fibrosis (n = 5 per group) induced by aging. (A) HW/BW heart weight/body weight. (B) LV/BW, left ventricular weight/body weight. Representative photomicrographs showing (C) nuclear volume and (D) collagen staining in the LV myocardial tissue, respectively (magnification ×40; scale bar: 250 μm). One-way ANOVA and Newman–Keuls post hoc were applied to detect differences between the groups. Significance level for the comparisons is shown above the bars. HW/BW = heart weight/body weight; LV/BW = left ventricular weight/body weight; YNT = young untrained; ONT = old untrained; OTR = old trained. Figure 2. View largeDownload slide Effects of swimming training on cardiac mass (YNT, n = 10; ONT, n = 9; OTR, n = 10) and fibrosis (n = 5 per group) induced by aging. (A) HW/BW heart weight/body weight. (B) LV/BW, left ventricular weight/body weight. Representative photomicrographs showing (C) nuclear volume and (D) collagen staining in the LV myocardial tissue, respectively (magnification ×40; scale bar: 250 μm). One-way ANOVA and Newman–Keuls post hoc were applied to detect differences between the groups. Significance level for the comparisons is shown above the bars. HW/BW = heart weight/body weight; LV/BW = left ventricular weight/body weight; YNT = young untrained; ONT = old untrained; OTR = old trained. Swimming Training Effects on LV Morphology and Performance Body weight was similar among the groups at the end of the study. Older rats showed suitable LV morphology alterations only on transthoracic echocardiography (Table 1). In this regard, an increased LV systolic area was the only modification compared to young animals, in which it was not observed upon training. Aging and exercise training had no impact on the LV performance on echocardiographic analysis. Hemodynamic measurements (Table 1) showed that LV end-diastolic pressure was significantly higher in older rats compared with the YNT group. Exercise training did not affect LV end-diastolic pressure. Table 1. Influence of Aging and Exercise Training on LV Morphology and Function   YNT (n = 10)  ONT (n = 9)  OTR (n = 10)  Body weight (g)  391 ± 13  415 ± 27  427 ± 22  Echocardiography   HR (bpm/min)  255 ± 12  247 ± 19  240 ± 20   LVDA (cm2)  0.49 ± 0.002  0.55 ± 0.02  0.51 ± 0.02   LVDA/BW (cm2/g)  0.0012 ± 0.0006  0.0013 ± 0.0006  0.0012 ± 0.0009   LVSA (cm2)  0.12 ± 0.01  0.18 ± 0.01*  0.15 ± 0.01   LVSA/BW (cm2/g)  0.00030 ± 0.00003  0.00042 ± 0.000027*  0.00035 ± 0.000034   FAC (%)  75 ± 1  68 ± 2  70 ± 3   E wave (cm/s)  0.64 ± 0.04  0.53 ± 0.03  0.62 ± 0.03   A wave (cm/s)  0.35 ± 0.02  0.31 ± 0.03  0.35 ± 0.01   E/A ratio  1.84 ± 0.12  1.8 ± 0.17  1.81 ± 0.13  Hemodynamic   HR (bpm/min)  349 ± 17  389 ± 4  366 ± 11   LVSP (mmHg)  119 ± 3  123 ± 6  127 ± 5   LVEDP (mmHg)  2.4 ± 0.2  3.7 ± 0.2*  4.7 ± 0.4*   +dP/dt (mmHg/s)  8,101 ± 624  9,340 ± 680  8,175 ± 752   −dP/dt (mmHg/s)  6,603 ± 390  5,811 ± 574  5,926 ± 321    YNT (n = 10)  ONT (n = 9)  OTR (n = 10)  Body weight (g)  391 ± 13  415 ± 27  427 ± 22  Echocardiography   HR (bpm/min)  255 ± 12  247 ± 19  240 ± 20   LVDA (cm2)  0.49 ± 0.002  0.55 ± 0.02  0.51 ± 0.02   LVDA/BW (cm2/g)  0.0012 ± 0.0006  0.0013 ± 0.0006  0.0012 ± 0.0009   LVSA (cm2)  0.12 ± 0.01  0.18 ± 0.01*  0.15 ± 0.01   LVSA/BW (cm2/g)  0.00030 ± 0.00003  0.00042 ± 0.000027*  0.00035 ± 0.000034   FAC (%)  75 ± 1  68 ± 2  70 ± 3   E wave (cm/s)  0.64 ± 0.04  0.53 ± 0.03  0.62 ± 0.03   A wave (cm/s)  0.35 ± 0.02  0.31 ± 0.03  0.35 ± 0.01   E/A ratio  1.84 ± 0.12  1.8 ± 0.17  1.81 ± 0.13  Hemodynamic   HR (bpm/min)  349 ± 17  389 ± 4  366 ± 11   LVSP (mmHg)  119 ± 3  123 ± 6  127 ± 5   LVEDP (mmHg)  2.4 ± 0.2  3.7 ± 0.2*  4.7 ± 0.4*   +dP/dt (mmHg/s)  8,101 ± 624  9,340 ± 680  8,175 ± 752   −dP/dt (mmHg/s)  6,603 ± 390  5,811 ± 574  5,926 ± 321  Note: Echocardiographic and hemodynamic analysis of left ventricular (LV) at the end of the study (YNT, n = 6; ONT, n = 9; OTR, n = 9). YNT = young untrained; ONT = old untrained; OTR = old trained; LV = left ventricule; LVDA = LV diastolic area; LVSA = LV systolic area; FAC = fractional area change; E/A ratio = ratio between the E and A waves; HR = heart rate; LVSP = LV systolic pressure; LVEDP = LV end-diastolic pressure; +dP/dt = maximal positive time derivate of developed pressure; −dP/dt = maximal negative derivate of developed pressure; BW = body weight. Data were analyzed with one-way ANOVA and post hoc Newman–Keuls test. *Different from the YNT group. View Large Swimming Training Improvements the Myocardial Mechanics in Older Animals There were remarkable differences in myocardial performance between the three groups. We observed that older rats had myocardial muscle that developed less force than their respective young controls; the result is depicted as a smaller DT and +dT/dt (Figure 3A and B). Moreover, −dT/dt was also significantly lower in older animals (Figure 3C). The myocardial mechanics was significantly affected by exercise training. Indeed, papillary muscles from the OTR group exhibited similar DT and +dT/dt data compared with the YNT group, as well as an improved −dT/dt. Figure 3. View largeDownload slide In vitro myocardial performance of the papillary muscle evaluated at lengths corresponding to 100% of Lmax (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Typical tracings representing mechanical parameters of the papillary muscle. (B) Peak developed tension (DT). Maximal (C, +dT/dt) positive and (D, −dT/dt) negative time derivative of DT. (E) Straight lines were fitted to the developed length–tension relationships using linear regression analysis. Mean DT slopes were compared between groups with one-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars (right graph). (F) Potentiation of post-rest contractions is evident in young untrained (YNT) rats while post-rest steady-state of force was reported in muscles of older rats with no effect of exercise training. Two-way repeated ANOVA and Bonferroni post hoc test was applied to detect differences between the groups and times. Significance level for comparisons between pause is shown above the bars (right graph). YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group for the corresponding pause. Figure 3. View largeDownload slide In vitro myocardial performance of the papillary muscle evaluated at lengths corresponding to 100% of Lmax (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Typical tracings representing mechanical parameters of the papillary muscle. (B) Peak developed tension (DT). Maximal (C, +dT/dt) positive and (D, −dT/dt) negative time derivative of DT. (E) Straight lines were fitted to the developed length–tension relationships using linear regression analysis. Mean DT slopes were compared between groups with one-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars (right graph). (F) Potentiation of post-rest contractions is evident in young untrained (YNT) rats while post-rest steady-state of force was reported in muscles of older rats with no effect of exercise training. Two-way repeated ANOVA and Bonferroni post hoc test was applied to detect differences between the groups and times. Significance level for comparisons between pause is shown above the bars (right graph). YNT = young untrained; ONT = old untrained; OTR = old trained. *p < .05 vs YNT group for the corresponding pause. As shown in Figure 3D, the DT was plotted as a dependent of muscle length. The active length–DT relationship from the ONT group was shifted downwards, with a lower slope value of linear worsened when compared to the other groups. Notably, the slope was preserved in exercise-trained rats. In this way, it is possible to state that for the same variation of muscle length, recruitment of the Frank–Starling mechanism is preserved in the older trained myocardium. In Figure 3E, there are post-rest contraction data that allow indirect evaluation of the SR function (16). In the healthy myocardium of rats, post-rest contraction is potentiated due to additional Ca2+ accumulated in the SR during the stimulus pause depending on SERCA2 activity and increased fractional Ca2+ release upon activation. Thus, the post-rest contraction was well evident in the YNT group, which increased with the duration of the pause. However, in the muscles from older groups, post-rest contraction behavior was changed. There was a plateau of the muscle force after the first stimulus pause, in which it was independent of the exercise training action. Swimming Training Modulates Expression of Ca2+-Regulating Proteins and Normalizes Myocardial Isoform Switches Changes in expression of proteins that modulate calcium handling have been a common finding in the older myocardium (17). In this study, older rats showed a significant decrease in NCX, SERCA2a, PLB-Thr17, and PLB-Ser16 expression (Figure 4). Furthermore, ONT animals exhibited higher L-Ca2+ content. Exercise training attenuated effects of aging on L-Ca2+, PLB-Thr17, and PLB-Ser16 expression. We also assessed whether older animals could have altered protein expression of α- and β-MHC (Figure 4I and J). These analyses were carried out because the distribution of α- and β-MHC showed to be directly correlated with myocardial dysfunction in aged tissue (18). There was no differential expression of α-MHC between the experimental groups (Figure 4I). However, Figure 4J illustrates that β-MHC expression was upregulated in the ONT group and unaltered in the OTR group. Therefore, β/α ratio was higher in the ONT group (Figure 4K) and preserved in trained animals. Figure 4. View largeDownload slide Myocardial protein expression evaluated by immunoblotting (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Representative blots for proteins that regulate the Ca2+ handling. The evaluated proteins were (B) Na+/Ca2+ exchanger (NCX), (C) L-type calcium channel (L-Ca2+), (D) ryanodine receptor (RyR), (E) sarcoplasmic reticulum Ca2+-ATPase (SERCA 2a), (F) phospholamban (PLB), (G) phospho-Thr17-PLB (PLB-Thr17), and (H) phospho-Ser16-PLB (PLB-Ser16). Protein expression of α-MHC and β-MHC isoforms is shown in I and J, respectively. (K) β-MHC/α-MHC ratio. All values were normalized for levels of GAPDH. One-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars. YNT = young untrained; ONT = old untrained; OTR = old trained. Figure 4. View largeDownload slide Myocardial protein expression evaluated by immunoblotting (YNT, n = 10; ONT, n = 9; OTR, n = 10). (A) Representative blots for proteins that regulate the Ca2+ handling. The evaluated proteins were (B) Na+/Ca2+ exchanger (NCX), (C) L-type calcium channel (L-Ca2+), (D) ryanodine receptor (RyR), (E) sarcoplasmic reticulum Ca2+-ATPase (SERCA 2a), (F) phospholamban (PLB), (G) phospho-Thr17-PLB (PLB-Thr17), and (H) phospho-Ser16-PLB (PLB-Ser16). Protein expression of α-MHC and β-MHC isoforms is shown in I and J, respectively. (K) β-MHC/α-MHC ratio. All values were normalized for levels of GAPDH. One-way ANOVA and Newman–Keuls post hoc test was applied to detect differences between the groups, and significance level for comparisons is shown above the bars. YNT = young untrained; ONT = old untrained; OTR = old trained. Discussion The current study has three main findings. First, swimming training-induced myocardial hypertrophy, and cardiomyocyte growth has been accompanied by a protective exercise effect on the myocardial structure. Thus, an increased interstitial collagen content was not present in trained older rats. Second, swimming training attenuated the effect of aging on several key proteins that modulate cardiomyocyte Ca2+ handling. A protective effect of exercise was most pronounced on β-MHC expression and β-MHC/α-MHC ratio, in which swimming training has mitigated increases evoked by aging. Third, older rats did not show substantial LV dysfunction but aging impaired the myocardial inotropism. Swimming training reversed the negative effect of aging, improving myocardial inotropism to a level similar to that of young rats. As illustrated in Figure 1, improved functional fitness was not identified when considering the VO2peak, which is a gold standard marker to assess exercise tolerance (19). On the other hand, when exercise performance was analyzed using a swimming approach, we identified a minor aerobic resistance in untrained older rats (~17%), while trained older rats showed a marked improvement (~50%). It is difficult to understand the disagreement between VO2peak and exhaustive swim test, but the training modality may have influenced the VO2peak measurement. Koch and colleagues (20) showed that maximal oxygen uptake increased more with aerobic interval training relative to continuous moderate exercise. In addition, the exercise training approach may have influenced our analysis, thereby, since the exercise training was carried out in the pool, the swimming test was more suitable to identify the improvement of physical fitness. Thus, a swimming test can be a useful tool to determine the physical fitness level of aging. Our results corroborate previous data that demonstrated myocardial hypertrophy in aged rats (21,22), which was more pronounced with exercise. Exercise training also had a significant effect on collagen concentration, where values in older trained rats were like those seen in young rats. Although other researchers showed similar findings, some data have been inconsistent. Thomas and colleagues (23) noted an increase in hydroxyproline LV concentration comparing 10 weeks with 22-months-old rats that was somewhat attenuated in the senescent rats who were submitted to a progressive treadmill incline (15%) at approximately 70% of maximal oxygen uptake (45 min/day; 5 days/week) for 10 weeks. However, Tomanek and colleagues (24) failed to find a significant collagen reduction in 22-months-old rats because of a motor-driven treadmill 6 days per week for a 12-week period. The no-singular findings may be due to species variances, maturational status, or even differences in exercise protocols. We have applied echocardiography and hemodynamic approaches to evaluate LV performance, and results from these experiments have revealed an only significant change in LVEDP, previous corroborating evidence that cardiac compliance decreases with aging (25,26). It is conceivable that the age-related decline in the compliance is likely a greater myocardial stiffness, and our findings of increased collagen LV concentration show to be a main cause of the altered stiffness in nontrained rats (23,25). However, LVEDP levels reached from 3.7 to 4.7 mmHg in elderly rats, which has been considered similar to the normal range for health young rats (27). Although LV performance has not been extensively altered in older rats, myocardial performance was worsened as assessed by both DT and +dT/dt on in vitro papillary muscle preparations. Moreover, aging evoked a downwards shift of resting length–DT curves, suggesting that the Frank–Starling relationship is also worsened (15). Lastly, aging may change the regular post-rest potentiation to post-rest stabilization of force. In fact, as most of the Ca2+ returns to the SR during rest, its content tends to accumulate with prolonged pause time, also increasing Ca2+ release and force generation in next activation (16). Hence, post-rest potentiation alterations may be attributed to malfunctioning SR, in which it converges in a relative reduction of free Ca2+. Our experiments suggest that impaired SR reuptake function may be due to decreased SERCA2a content and its regulatory proteins (PLB-Thr17/Ser16). Moreover, altered SERCA2a and PLB-Thr17/Ser16 also helps explain slowed myocardial relaxation (−dT/dt) in older rats. An interesting aspect of this study is the reduced NCX and increased L-Ca2+ myocardial content, which to our knowledge is a finding described only by few investigations (28). The repercussion of these discoveries of muscle function shown to be clarified, but a first step could be to investigate the impact of altered NCX and L-Ca2+ on Ca2+ handling because a prolonged Ca2+ transients (3) or slower Ca2+ transient decay arises (17) are commonly reported in older rats. Finally, our findings corroborate that age-related change in contractile proteins is an upright shift of slower β-MHC, which can slow relaxation (17). Moreover, increased β-MHC expression may also partially explain lower +dT/dt of aged papillary muscles. Studies carried out on myofibrillar ATPase activity have reported that the speed with which muscle shortens is correlated with the ATP-hydrolyzing capacity of the myosin (18). The most important finding of the present study was the effectiveness of swimming training on improvement of myocardial contractility in older rats. Exercise resulted in maintenance of myocardial inotropism and contraction speed as well as improvement of relaxation. Moreover, Frank–Starling mechanism was also preserved in trained aged rats. To our knowledge, previous studies have examined the role of running exercise training in myocardial performance, in which the duration of training has varied between 4 and 20 weeks on a rodent treadmill. These studies have identified benefits (3,4) or not (5,6) of exercise training on contractile indices of myocardial performance (eg, longer time-to-peak tension). In our study, an increased rate of contraction (+dT/dt) and relaxation (−dT/dt) myocardial was accompanied by improved inotropism (DT) in older rats undergoing 6 weeks swimming training. These results suggest that better results for myocardial mechanics can be obtained with a swimming training program compared to running in rats. The underlying mechanism for the improvement in contractility is said to be multifactorial, including increased myocardial catecholamine stores (4), improvement in metabolic enzyme activity (29) as well as mitochondrial function (30), maintenance of myocyte number because of the anti-apoptotic exercise effect (31), and increase in atrial myosin light chain 1 expression (32). In our study, we have identified other mechanisms that may be involved in the improvement of exercise-induced myocardial performance. Inhibition of age-related changes in collagen content could help to explain the exercise-induced improvement of myocardial relaxation. We also provided evidence that intrinsic cardioprotective benefits in older rats can be associated with changes in several proteins that modulate Ca2+ handling. Maintenance of PLB-Ser16 levels and improvement of the PLB-Thr17 content may be associated with functional SERCA2a improvement. These findings may explain an increased calcium transport by the SERCA, in which it shows potential mechanisms underlying the improvement of myocardial performance with exercise training initiated during senescence (3). Notwithstanding, the preserved β-MHC/α-MHC ratio could be involved in the maintenance of myocardial shortening and relaxation speed as well as conserved Frank–Starling mechanisms in trained older rats. In summary, swimming training protected age-induced remodeling of myocardial tissue. It includes increased connective tissue and attenuated myocardial performance. Regular exercise also improved key proteins that regulate the Ca2+ handling and sarcomere velocity. It should be noted, however, that in the current design, it cannot be determined whether the benefits of exercise training are more evident to counteract aging over the longer term, in which exercise could be started in middle-aged animals to avoid adverse myocardial effects noticed in older animals. However, our study indicates that exercise is useful to improve myocardial mechanics when initiated in senescence. Although exercise resulted in improvement of myocardial inotropism associated with attenuation of the effect of aging on proteins that modulate intracellular calcium (LCa2+, PLB-Thr17, and PLB-Ser16), we were unable to directly test whether exercise training exercise modulates cardiomyocyte calcium handling in the older rats. Funding This work was supported by the National Council for Scientific and Technological Development (CNPq; 479395/2012–8) and the São Paulo Research Foundation (FAPESP; 15/11028–9 and 13/10619-8). Acknowledgments We thank Editage Translation Company for critical reviewing of English language. Conflict of Interest None declared. References 1. Floegel TA, Perez GA. An integrative review of physical activity/exercise intervention effects on function and health-related quality of life in older adults with heart failure. 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The Journals of Gerontology Series A: Biomedical Sciences and Medical SciencesOxford University Press

Published: Apr 1, 2018

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