ORIGINAL ARTICLE

Effects of Endurance Training on Myocardial Hypertrophy and Ventricular Function in a Transgenic Mouse Model with sympathetic Hyperactivity

Efectos del ejercicio intenso sobre la hipertrofia miocárdica y la función ventricular en un modelo de ratón transgénico con hiperactividad simpática

 

LUCIANA WILENSKY, NADIA L. MART ÍNEZ NAYA, JAZMIN KELLY, PABLO CASSAGLIA, MARÍA E. ARUANNO, BRUNO BUCHHOLZ^MTSAC, ISAAC L. MOGUNOVSKY MICHELL, CELINA MORALES^MTSAC, GERMÁN E. GONZÁLEZ

 

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ABSTRACT

Background: Previous studies have shown that endurance training (ET) reduces inotropic, chronotropic and lusitropic reserve in normal mice.
Objective: The aim of this study was to evaluate the effect of endurance training on the inotropic and chronotropic reserve of trans-genic mice with sympathetic hyperactivity induced by overexpression of the cardiac GSα protein.
Methods: Endurance training consisted in two daily 90-min sessions, 6 days/week, during 4 weeks. Four experimental groups were formed: 1) non-transgenic sedentary (nonTG Sed); 2) transgenic sedentary (TG Sed); 3) nonTG+ET and 4) TG+ET.
Results: Endurance training induced myocardial hypertrophy [left ventricular weight (g)/tibial length (mm)] from 5.3±0.3 and 5.5±0.2 in nonTG Sed and TG Sed to 6.8±0.1 and 6.8±0.3 in nonTG+ET and TG+ET, respectively (p<0.05 nonTG Sed vs. nonTG+ET and TG Sed vs. TG+ET). Isoproterenol administration (56 ng/kg) increased +dP/dtmax by 63±10% in nonTG Sed (p<0.05 vs. baseline), 34±2% in TG Sed (p<0.05 vs. baseline and p<0.05 vs. nonTG Sed), 36±7% in non TG+ET (p<0.05 vs. base-line) and 36±7% in TG+ET (p<0.05 vs. baseline). Heart rate (beats/min) increased from 301±15 to 528±37 in nonTG Sed (p<0.05 vs. baseline), from 519±57 to 603±41 in TG Sed, from 300±16 to 375±20 in nonTG+ET (p<0.05 vs. baseline) and from 484±18 to 515±21 in TG+ET. Interstitial collagen was similar among groups.
Conclusions: These results suggest that endurance training decreases inotropic and chronotropic reserve without generating struc-tural changes associated to pathological hypertrophy. The presence of sympathetic hyperactivity does not modify this response.

Key words: Exercise Tolerance - Physical Conditioning, Animal - Sympathetic Nervous System - Isoproterenol

RESUMEN

Introducción: En estudios previos mostramos que el ejercicio intenso (EI) reduce la reserva inotrópica, cronotrópica y lusitrópica en ratones normales.
Objetivo: Evaluar el efecto del ejercicio intenso sobre la reserva inotrópica y cronotrópica en un modelo de ratones transgénicos con sobreexpresión cardíaca de la proteína Gsα, que induce un cuadro de hiperactividad simpática.
Material y métodos: El ejercicio consistió en dos sesiones diarias de 90 minutos de natación, 6 días/semana durante 4 semanas. Se utilizaron cuatro grupos experimentales: 1: sedentario no transgénico (noTG Sed); 2: sedentario TG (TG Sed); 3: noTG+EI y 4: TG+EI.
Resultados: El ejercicio indujo el desarrollo de hipertrofia miocárdica [índice peso del ventrículo izquierdo (g)/longitud de la tibia (mm)] desde 5,3±0,3 y 5,5±0,2 en noTG Sed y TG Sed a 6,8±0,1 y 6,8±0,3 en noTG+EI y TG+EI, respectivamente (p<0,05 noTG Sed vs. noTG+EI y TG Sed vs. TG+EI). La administración de isoproterenol (56 ng/kg) incrementó la +dP/dtmáx 63% ±10% en noTG Sed (p<0,05 vs. basal); 34% ±2% en TG Sed (p<0,05 vs.basal y p< 0,05 vs. noTG Sed); 36% ±7% en noTG+EI (p<0,05 vs. basal) y 36% ±7% en TG+EI (p<0,05 vs.basal). La frecuencia cardíaca aumentó de 301±15 a 528±37 latidos/min en noTG Sed (p<0,05 vs. basal), de 519±57 a 603±41 latidos/min en TG Sed, de 300±16 a 375±20 en noTG+EI (p<0,05 vs. basal) y de 484±18 a 515±21 en TG+EI. El colágeno intersticial fue similar entre los grupos.
Conclusiones: Estos resultados sugieren que el ejercicio intenso disminuye la reserva inotrópica y cronotrópica sin generar cambios estructurales vinculados a la hipertrofia patológica. La presencia de hiperactividad simpática no modifica esta respuesta.

Palabras clave: Tolerancia al ejercicio - Condicionamiento físico animal - Sistema nervioso simpático - Isoproterenol

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Abbreviations

beats/min Beats per minute
+dP/dtmáx Maximum time derivative of pressure
ET Endurance training
HR Heart rate
ISO Isoproterenol
LV Left ventricle
mRNA Messenger ribonucleic acid
nonTG Non transgenic
RV Right ventricle
Sed Sedentary
TG Transgenic
TL Tibial length

 

INTRODUCTION

Different studies have shown that regular physical activity prevents cardiac diseases and that chronic exercise attenuates the main cardiovascular risk fac-tors. (1) Depending on the type, exercise can be as-sociated with mild cardiac dilation, which represents a favorable adaptation of the heart to compensate increased functional demand, allowing it to preserve or even increase ventricular function. (2, 3) Previous studies indicated that low-intensity exercise may de-lay the development of heart failure and improve survival in spontaneously hypertensive rats, suggesting that moderate to mild exercise has beneficial effects. (4) However, experimental protocols of chronic endur-ance training (ET) performed in rats, showed that the animals developed cardiac hypertrophy with patho-logic characteristics, increasing pro-fibrotic markers, right ventricular fibrosis, arrhythmia susceptibility and ventricular dysfunction. (5) In this sense, despite exercise is recommended as an efficient therapeutic strategy for different cardiovascular pathologies, the intensity/response relationship is still poorly under-stood and the mechanisms have not been fully investi-gated. (6) We have previously shown that ET reduces inotropic, chronotropic and lusotropic reserve in normal mice. (7) Since sympathetic activity is part of the physiology of exercise, the aim of this study was to analyze whether the decrease in inotropic and chrono-tropic reserve produced by ET was present in a trans-genic model of sympathetic hyperactivity submitted to an ET protocol.

METHODS

Male, 3-month-old mice (31±1 g) corresponding to the non-transgenic FVB strain (nonTG) and the TG strain overex-pressing the specific cardiac Gsα protein were used. Gsα protein overexpression was achieved through the insertion of the sequence contiguous to the promoter gene of the α-myosin heavy chain expressed in cardiomyocytes, using the lines showing 38-fold mRNA increase and 2.8 elevation of Gsα protein content. This genetic modification is pheno-typically evident by the characteristic sympathetic hyper-activity of the model, described as a significant increase of heart rate and contractility. (8, 9)

Experimental protocol

An intense exercise protocol consisting of two daily 90-min sessions, 6 days a week, was performed during 4 weeks. Mice were placed in a 40×70×30 cm pool with water maintained at 30-32 ºC via a temperature stabilizer. (10) At the begin-ning of the protocol, the animals were adapted for a week, starting with a 20-minute swimming period which was extended in the successive days to reach the complete protocol time. (11-13) The animals were housed at 20-22 ºC with 12 h light/darkness cycles and ad libitum food and water availability.

Experimental groups

Four experimental groups were formed: 1) Control nonTG sedentary group (n=14): animals did not perform exercise and were kept in their respective cages until euthanasia; 2) Control TG sedentary group (n=5): animals did not perform exercise and were kept in their respective cages until eutha-nasia; 3) nonTG+ET (n=14): animals performed the ET protocol previously described, and 4) TG+ET (n=16): TG mice performed the ET protocol previously described.

After completion of the protocol period all the experimental groups underwent in vivo baseline ventricular func-tion and inotropic and chronotropic reserve studies followed by complete autopsy.

In vivo ventricular function studies

After completion of the protocol period, the nonTG (n=8), TG (n=3), nonTG+ET (n=9) and TG+ET (n=8) groups were weighed and anesthetized with ketamine (100 mg/kg) and xy-lazine (2.5 mg/kg). The right carotid artery was dissected and a heparinized catheter was inserted and advanced into the left ventricle (LV). The left jugular vein was also dissected and another catheter was inserted for intravenous bolus injection of isoproterenol (ISO, 56 ng/kg). After stabilization (10 minutes), baseline left ventricular pressure, its first derivative (+dP/dt_max, mmHg/s) and heart rate (HR, beats/min) were re-corded. (14) The same variables were recorded for each group after ISO administration, using a computer equipped with an analog to digital converter (National Instruments) and software for data acquisition and analysis. (15)

assessment of cardiac hypertrophy

After the ventricular function study, the animals were sacri-ficed and the corresponding autopsy was done. The complete cardiopulmonary block was removed and the LV, the RV and both atria were dissected and weighed. The LV was fixed in formaldehyde buffer for later inclusion in paraffin and stain-ing for collagen quantification. Body weight was also record-ed before and after the exercise protocol and tibial length (TL) was measured to calculate hypertrophy coefficients (LV/body weight (BW) and LV/TL). (16) Both indexes were considered and compared, as BW evidenced great variability with training; however, TL is a growth index that does not change with exercise.

Quantifcation of interstitial collagen

Interstitial collagen was quantified with colorimetric tech-nique in histological sections of nonTG (n=5), TG (n=3), nonTG+ET (n=7) and TG+ET (n=7) groups stained with picrosirius red, using Image-Pro Plus 6.0 digital image ana-lyzer. Results were expressed as percent collagen in the total LV per field. Perivascular collagen was not considered in any group. (17)

statistical analysis

Data are expressed as mean±standard error of the mean. Sigma STAT32 software package was used for data statisti-cal analyses and ANOVA followed by Bonferroni to compare among groups. A p value <0.05 was considered statistically significant.

Ethical considerations

All the experiments were performed according to the Na-tional Institute of Health "Guide for the Care and Use of Laboratory Animals" (NIH Publication 85-3, revised 1985).

RESULTS

All the animals that started finished the exercise pro-tocol without any deaths from exhaustion or other causes.

Endurance training significantly increased the my-ocardial hypertrophy index similarly in both groups (nonTG+ET and TG+ET) (Table 1).

In the in vivo ventricular function study, we con-firmed that baseline HR (Figure 1) and myocardial contractility (+dP/dt_max, Figure 2) were significantly increased in TG animals (sedentary (Sed) and exer-cise), in response to the sympathetic hyperactivity of the experimental model.

Heart rate in nonTG (nonTG Sed: 301±15 vs. nonTG+ET: 300±16 beats/min) as in TG mice sub-

mitted to exercise was not modified with respect to their corresponding baseline valúes (TG Sed: 519±57 vs. TG+ET: 484±18 beats/min) (Figure 1A). Isopro-terenol administration only increased HR in nonTG groups (nonTG Sed: from 301±15 to 528±37 beats/ min, and nonTG+ET: from 300±16 to 375±20 beats/ min, p<0.05), with no signifícant changes in TG groups (TG Sed: from 519±57 to 603±41 beats/min and TG+ET: from 484±18 to 515±21 beats/min). The percent increase in HR (Figure IB) revealed a signifícantly lower response in TG Sed, nonTG+ET and TG+ET groups compared with the nonTG Sed group (TG Sed: 13.7%±4.5%; nonTG+ET: 27.7%±5.3 %; TG+ET: 9.9%±1.3%, p<0.05).

Exercise did not modify baseline contractile state, assessed through +dP/dt_max, in nonTG (nonTG Sed: 5315±382 mmHg/s, nonTG+ET: 6350±531 mmHg/s), ñor in TG (TG Sed: 7681±513 mmHg/s, TG+ET: 7495±219 mmHg/s) groups (Figure 2). Myocardial contractility did not increase signifícantly with ISO administration in all the experimental groups (nonTG Sed: from 5314±382 mmHg/s to 9218±605 mmHg/s; TG: from 7681±513 mmHg/s to 10084±421 mmHg/s; nonTG+ET: from 6350±531 mmHg/s to 8128±374 mmHg/s and TG+ET: from 7495±219 mmHg/s to 10226±679 mmHg/s; p<0.05 for all cases), although the percent increase was different among groups, with a significant decrease in the response to ISO in TG Sed (34%±2%), nonTG+ET (36%±7%) and TG+ET (36%±7%) groups, compared with nonTG Sed (63%±10%, p<0.05 vs. all the other groups) (Figure 2B).

 

 

Interstitial collagen was similar in all the experimental groups (nonTG Sed: 2.25%±0.30%, TG Sed: 2.50%±0.4%, nonTG+ET: 2.46%±0.30% and TG+ET: 2.65%±0.54%) (Figure 3).

DISCUSSION

The present study showed that ET in normal mice, as well as in those with specific cardiac Gsα overex-pression produces a significant impairment of ino-tropic and chronotropic reserve with no changes in interstitial collagen. Moreover, results showed that the decreased inotropic reserve in normal animals was similar to that seen in both TG groups, suggest-ing that the effect of ET did not enhance the decrease of contractile reserve in TG mice. In agreement with previous studies (4, 5, 7, 10), no deaths from exertion were observed in the groups that performed ET, prob-ably due to the previous week of adaptation before starting the protocol, during which they got used to the environment and improved their aerobic capacity. In previous studies we showed that ET decreas-es inotropic, chronotropic and lusotropic reserve in mice. (7) The present work extends these findings analyzing ET-induced inotropic and chronotropic reserve impairment in a TG model with specific cardiac sympathetic hyperactivity. Thus, the present study showed that overexpression of the cardiac protein Gsα increases HR as well as contractility in baseline condi-tions. However, these animals developed myocardial hypertrophy secondary to ET, similarly to nonTG ani-mals. Also, a decreased inotropic and chronotopic reserve in nonTG+ET mice was observed, not generat-ing the expected cardioprotection. Moreover, TG+ET animals preserved their functional parameters compared with their sedentary control (TG Sed), as these started from higher values than the nonTG Sed group, without modification of the values in response to beta-adrenergic stimulation.

Fig. 3. Interstitial collagen assessed in histological sections stained with picrosirius red in all the experimental groups. There were no signifcant differences in all the groups studied. LV: Left ventricular. nonTG: Non transgenic. TG: Transgenic. Sed: Sedentary. ET: Endurance training.

 

In the in vivo ventricular function assessment, the changes in HR could modify contractility, and since the TG model has a significant increase in HR, these data should be confirmed evaluating ventricular func-tion and inotropic reserve in an in vitro model allow-ing a strict control of variables, as for example con-stant HR, ventricular volume and coronary flow. (18, 19)

In our experimental conditions, the hearts of ani-mals that were submitted to exercise (nonTG and TG) developed myocardial hypertrophy without fibro-sis, suggesting that, at least from a structural point of view, exercise produced adaptive hypertrophy. (6)

However, while it is well known that exercise activates the sympathetic nervous system, (20) the myocardial response to different levels of physical activity in the presence of sympathetic stimulation has not been identified. Our data suggest that although the TG model shows increased contractility and HR, this does not produce a greater deleterious effect, which is oth-erwise observed in nonTG animals, where a decreased inotropic and chronotropic reserve outweighs the ben-eficial effect of exercise.

CONCLUSIONS

Our results indicate that ET comparable to beta-adr-energic stimulation decreases in a non-additive way the contractile reserve without generating structural changes associated to maladaptive hypertrophy.

Conficts of interest

None declared. (See authors' conflicts of interest forms in the website/Supplementary material).

REFERENCES

1. Powers SK, Lennon SL, Quindry J, Mehta JL. Exercise and cardio-protection. Curr Opin Cardiol 2002;17:495-502. http://doi.org/dzczzv

2. Prior DL, La Gerche A. The athlete's heart. Heart 2012;98:947-55. http://doi.org/737

3. Rawlins J, Bhan A, Sharma S. Left ventricular hypertrophy in athletes. Eur J Echocardiogr 2009;10:350-6.         [ Links ] http://doi.org/dnx9ph

4.  Garciarena CD, Pinilla OA, Nolly MB, Laguens R P, Escudero EM, Cingolani HE, Ennis IL. Endurance training in the spontane-ously hypertensive rat. Hypertension 2009;53:708-14. http://doi.org/ cwqvjk

5. Benito B, Gay-Jordi G, Serrano-Mollar A, Guasch E, Shi Y, Tardif JC, et al. Cardiac arrhythmogenic remodeling in a rat model of long-term intensive exercise training. Circulation 2011;123:13-22. http:// doi.org/dhgwsv

6. La Gerche A. Can intense endurance exercise cause myocardial damage and fibrosis? Curr Sports Med Rep 2013;12:63-9. http://doi. org/738

7.  Wilensky L, González GE, D'Annunzio V, Matorra F, Pérez V, Gullace FA y cols. Efectos del ejercicio intenso sobre la función ventricular basal y la respuesta inotrópica, cronotrópica y lusitrópica en ratones. Rev Argent Cardiol 2013;81:115-21. http://doi.org/739

8. Gaudin C, Ishikawa Y, Wight DC, Mahdavi V, Nadal-Ginard B, Wagner TE, et al. Overexpression of Gs alpha protein in the hearts of transgenic mice. J Clin Invest 1995;95:1676-83. http://doi.org/dnvf8x

9. Muntz KH, Ishikawa Y, Wagner T, Homcy CJ, Vatner S F, Vatner DE. Localisation of cardiac Gs alpha in transgenic mice overexpressing Gs alpha. J Mol Cell Cardiol 1997;29:1649-53. http://doi.org/frcq2f

10.  Evangelista FS, Brum PC, Krieger JE. Duration-controlled swimming exercise training induces cardiac hypertrophy in mice. Braz J Med Biol Res 2003;36:1751-9. http://doi.org/bvw42v

11. Ikeda H, Shiojima I, Ozasa Y, Yoshida M, Holzenberger M, Kahn CR, et al. Interaction of myocardial insulin receptor and IGF receptor signaling in exercise-induced cardiac hypertrophy. J Mol Cell Cardiol 2009;47:664-75. http://doi.org/d59mcj

12. Iemitsu M, Miyauchi T, Maeda S, Sakai S, Kobayashi T, Fujii N, et al. Physiological and pathological cardiac hypertrophy induce dif-ferent molecular phenotypes in the rat. Am J Physiol Regul Integr Comp Physiol 2001;281:R2029-36.

13. Kaplan ML, Cheslow Y, Vikstrom K, Malhotra A, Geenen DL, Nakouzi A, et al. Cardiac adaptations to chronic exercise in mice. Am J Physiol 1994;267:H1167-73.

14. González GE, Rabald S, Briest W, Gelpi RJ, Seropian I, Zimmer HG, et al. Ribose treatment reduced the infarct size and improved heart function after myocardial infarction in rats. Cell Physiol Bio-chem 2009;24:211-8. http://doi.org/dmjgjf

15. Marchini T, Magnani N, D'Annunzio V, Tasat D, Gelpi RJ, Álva-rez S, et al. Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter. Biochim Biophys Acta 2013;1830:2545-52. http://doi.org/74b

16. Kim J, Wende AR, Sena S, Theobald HA, Soto J, Sloan C, et al. Insulin-like growth factor I receptor signaling is required for exer-cise-induced cardiac hypertrophy. Mol Endocrin 2008;22:2531-43. http://doi.org/d8w7mb

17. Schwarz A, Godes M, Thone-Reineke C, Theuring F, Bauer C, Neumayer HH, et al. Tissue-dependent expression of matrix pro-teins in human endothelin-1 transgenic mice. Clin Sci (Lond) 2002;103(Suppl 48):39S-43S. http://doi.org/74c

18. González GE, Mangas F, Chauvin AD, Monroy S, Donato M, Morales C, et al. Diastolic behavior during post ischemic hypercontrac-tion phase in rabbit stunned myocardium. Medicina 2003;63:403-9.

19. González GE, Rodriguez M, Donato M, Palleiro J, D'Annunzio V, Morales C, et al. Effects of low-calcium reperfusion and adenos-ine on diastolic behavior during the transitory systolic overshoot of the stunned myocardium in the rabbit. Can J Physiol Pharmacol 2006;84:265-72. http://doi.org/cx7rs3

20. Ng J, Sundaram S, Kadish AH, Goldberger JJ. Autonomic effects on the spectral analysis of heart rate variability after exercise. Am J Physiol Heart Circ 2009;297:H1421-8. http://doi.org/dvbkqs