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Abnormal cardiac response to exercise in a murine model of familial hypertrophic cardiomyopathy
International Journal of Cardiology 119 (2007) 245 – 248 www.elsevier.com/locate/ijcard
Letter to the Editor
Abnormal cardiac response to exercise in a murine model of familial hypertrophic cardiomyopathy Lan Nguyen a , Jessica Chung a , Lien Lam a , Tatiana Tsoutsman a,b , Christopher Semsarian a,b,c,⁎ a
Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Australia b Faculty of Medicine, University of Sydney, Australia c Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia Received 8 September 2006; accepted 13 September 2006 Available online 24 October 2006
Abstract Clinical outcome in familial hypertrophic cardiomyopathy (FHC) may be influenced by modifying factors such as exercise. Transgenic mice which overexpress the human disease-causing cTnI gene mutation, Gly203Ser (designated cTnI-G203S), develop all the characteristic phenotypic features of FHC. To study the modifying effect of exercise in early disease, mice underwent swimming exercise at an early age prior to the development of the FHC phenotype. In non-transgenic and cTnI-wt mice, swimming resulted in a significant increase in left ventricular wall thickness and contractility on echocardiography, consistent with a physiological hypertrophic response to exercise. In contrast, cTnI-G203S mice showed no increase in these parameters, indicating an abnormal response to exercise. The lack of a physiological response to exercise may indicate an important novel mechanistic insight into the role of exercise in triggering adverse events in FHC. Crown Copyright © 2006 Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Hypertrophy; Cardiomyopathy; Exercise; Troponin I gene
1. Introduction Familial hypertrophic cardiomyopathy (FHC) is a primary disorder of the myocardium characterised by cardiac hypertrophy in the absence of other loading conditions such as hypertension [1]. Vigorous exercise in FHC has been associated with sudden cardiac death, particularly in the young, and is the basis for the recommendation to FHC patients to avoid competitive sports [2,3]. The mechanistic link between exercise and sudden death in FHC may relate to alterations in Ca2+ homeostasis within cardiomyocytes, changes in energy utilisation, or the histopathological changes, including myocyte disarray and interstitial fibrosis, characteristically seen in
FHC acting as a substrate for ventricular arrhythmias [4,5]. While much is known about how the normal heart responds to exercise, relatively less is known about how this response to exercise is altered in FHC. Since normal Ca2+ handling in cardiac cells is altered during exercise [6], disruptions to components of the sarcomere critical for responding to increases in intracellular Ca2+, e.g. mutations in the cardiac troponin I gene [7], may be important in understanding the association between exercise and sudden death in FHC. This study sought to evaluate the cardiac response to exercise in early (pre-hypertrophic) development of FHC. 2. Materials and methods
⁎ Corresponding author. Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Locked Bag 6, Newtown NSW 2042, Australia. Tel.: +61 2 9565 6195; fax: +61 2 9565 6101. E-mail address: [email protected] (C. Semsarian).
2.1. Mouse model and exercise protocol We have recently described the cTnI-G203S transgenic mouse model, which over-expresses the human disease-
0167-5273/$ - see front matter. Crown Copyright © 2006 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2006.09.001
246
L. Nguyen et al. / International Journal of Cardiology 119 (2007) 245–248
Fig. 1. Changes in lactate level (A) and heart:body weight (B) in exercised (grey bars) versus sedentary (black bars) mice. (⁎p b 0.001 versus sedentary mice; minimum n = 5 per group). Data expressed as mean ± SD. Proposed model for differential response to exercise in normal compared to FHC mice (C).
causing cTnI gene mutation, Gly203Ser, in a cardiac-specific manner [8]. These mice develop all the phenotypic features of FHC by age 20–50 weeks depending on the level of protein expression of the mutant gene [8]. Male mice (n = 10) aged 8– 10 weeks were swum in groups of 5 in a pool where the water was heated to 28–32 °C by a warming tray and maintained at this temperature throughout the swimming period. Mutant cTnI-G203S mice from a low transgene-expression line (later onset of FHC features) were compared to non-transgenic (NTG) mice, and those over-expressing wild-type troponin I (cTnI-wt), which are indistinguishable from NTG mice [8]. Mice were initially swum for 10 min twice daily and the duration increased by 10-min increments daily. Upon reaching 60 min twice daily, this duration was maintained for the remainder of the 4-week exercise period. Mice were monitored daily and weighed weekly. Blood lactate levels were measured to monitor exercise intensity levels. Age and strain matched non-exercised sedentary controls were maintained and handled daily, consistent with the daily handling of the swum mice. All mouse experiments were performed in strict accordance with the Institutional Animal Ethics Committee.
2.2. Echocardiography Mice were evaluated longitudinally using 2D and M-mode mouse echocardiography during the study period. Transthoracic echocardiography was performed using a 7–15 MHz linear array probe and a HDI 5000 ultrasonograph (Acuson) as previously described [9]. 2.3. Histopathology Hearts were excised, washed in 1 × PBS, blot dried, and weighed. Heart weight:body weight ratios were calculated for each mouse. Cardiac tissue was treated for histological examination as described previously [10]. In brief, excised and washed hearts were placed in 10% formaldehyde. Sections were fixed, embedded and tissues stained with either hematoxylin and eosin or Masson's Trichrome. 2.4. Statistical analysis All values are given as mean ± standard deviation. Unpaired t tests were used to determine significant
L. Nguyen et al. / International Journal of Cardiology 119 (2007) 245–248 Table 1 Echocardiographic characteristics of exercised mice Genotype/exercise
HR (bpm)
LVWT (mm)
FS (%)
NTG
502 ± 41 456 ± 25⁎ 531 ± 32 460 ± 30⁎ 512 ± 50 501 ± 24
0.90 ± 0.03 1.08 ± 0.04† 0.90 ± 0.03 1.08 ± 0.02† 0.95 ± 0.04 0.97 ± 0.03
66 ± 2 72 ± 3⁎ 66 ± 2 71 ± 1† 65 ± 3 67 ± 4
cTnI-wt cTnI-G203S
Sedentary Swim Sedentary Swim Sedentary Swim
Data expressed as mean ± SD. HR = heart rate, LVWT = maximal left ventricular wall thickness, FS = fractional shortening, NTG = non-transgenic. ⁎p b 0.001, †p b 0.05 versus sedentary mice of same genotype.
differences between exercised and sedentary mice within genotype groups. For all comparisons, a value of P b 0.05 was considered significant. 3. Results Mice were exercised for a period of 4 weeks by swimming and compared to non-exercised sedentary controls. No sudden deaths occurred during the course of these studies. Swum mice had reduced blood lactate levels (Fig. 1A) compared to non-exercised controls, consistent with chronic exercise. There was no difference in body weight between age-matched sedentary and exercised mice in all study groups over the exercise study period. NTG and cTnI-wt mice had a different cardiac response to exercise compared to the cTnI-G203S FHC mice. NTG and cTnI-wt mice showed a physiological response to exercise characterised by mild cardiac hypertrophy, increased contractile function, and a lower resting heart rate following their 4-week exercise program (Table 1). In contrast, cTnI-G203S mice demonstrated an absence of these physiological responses. Specifically, in NTG and cTnI-wt mice, exercise resulted in a significant increase in heart:body weight ratio (Fig. 1B). Mouse echocardiography showed that LV wall thickness was also increased with exercise, while heart rates in these mice were significantly lower than in non-exercised mice (Table 1). These findings are consistent with a physiological hypertrophic response to exercise. In contrast, cTnI-G203S mice showed no increase in heart:body weight ratio or left ventricular wall thickness on echocardiography, indicating an abnormal response to exercise (Table 1). LV systolic function was increased following the swimming program in NTG and cTnI-wt mice, but unchanged in swum cTnI-G203S mice (Table 1). No changes in histopathology were observed in exercised mice. 4. Discussion FHC mice demonstrate an abnormal response to exercise. NTG and cTnI-wt swum mice developed a normal physiological response to exercise, while cTnI-G203S mice lacked this adaptive response. Such an abnormal response to exercise may be an important substrate for adverse events in FHC associated with competitive exercise.
247
The association of competitive exercise with adverse events, including sudden death, remains a significant clinical problem in FHC. FHC remains the commonest structural cause of sudden cardiac death in those aged under 35 years, including competitive athletes [2,3]. Sudden death occurs either during, or immediately after exercise in up to 70% of patients. While ventricular arrhythmias are the most common cardiac arrhythmia preceding death, the triggering events and the nature of the arrhythmogenic substrate remain unclear. Furthermore, it is well established that in approximately 30% of FHC patients, an abnormal haemodynamic/blood pressure response to exercise is observed, which independently acts as an important prognostic risk indicator. The findings of the current study suggest one possibility for the adverse response to exercise seen in FHC may relate to an abnormal response to exercise whereby the normal physiological adaptations to exercise, including improved cardiac contractility, reduced heart rate, and physiological cardiac hypertrophy, are absent. After a 4-week strenuous swimming program, cTnI-G203S mice did not develop a normal physiological response to exercise as seen in NTG and cTnIwt littermates. Importantly, this failure of physiological adaptation to exercise occurred in cTnI-G203S mice at an age prior to the development of the phenotypic features of FHC, indicating the mutation alone is primarily responsible for the difference in exercise response observed and not secondary changes associated with developed pathological cardiac hypertrophy, diastolic impairment and histopathological changes. Abnormal calcium homeostasis, observed in these FHC mice [8] and in other FHC mouse models [9,10], may be involved in this abnormal response to exercise (unpublished data; Fig. 1C). The clinical heterogeneity seen in FHC, in which affected individuals within the same family, and therefore carrying the same mutation, can have vastly different clinical outcomes, indicates both environmental and genetic modifying influences are important mediators of disease and clinical outcome. Utilising various animal models harbouring different gene mutations, such at the cTnI-G203S model presented here, will allow both increased understanding of disease pathogenesis and to enable modifying factors such as exercise to be evaluated in FHC. Acknowledgements CS is the recipient of a National Health and Medical Research Council Practitioner Fellowship. The research is supported by project grants from the National Heart Foundation and the National Health and Medical Research Council of Australia. References [1] Chung J, Tsoutsman T, Semsarian C. Hypertrophic cardiomyopathy: from gene defect to clinical disease. Cell Res 2003;13:9–20. [2] Maron BJ, Shirani J, Poliac LC, Mathenge R, Roberts WC, Mueller FO. Sudden death in young competitive athletes-clinical, demographic and pathological profiles. JAMA 1996;276:199–204.
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L. Nguyen et al. / International Journal of Cardiology 119 (2007) 245–248
[3] Maron BJ. Sudden death in young athletes. N Engl J Med 2003;349:1064–75. [4] Morita H, Seidman J, Seidman CE. Genetic causes of human heart failure. J Clin Invest 2005;115:518–26. [5] Ashrafian H, Redwood C, Blair E, Watkins H. Hypertrophic cardiomyopathy: a paradigm for myocardial energy depletion. Trends Genet 2003;19:263–8. [6] Lehnart SE, Wehrens XH, Marks AR. Calstabin deficiency, ryanodine receptors, and sudden cardiac death. Biochem Biophys Res Commun 2004;322:1267–79. [7] Doolan A, Tebo M, Ingles J, et al. Cardiac troponin I mutations in Australian families with hypertrophic cardiomyopathy: clinical, genetic and functional consequences. J Mol Cell Cardiol 2005;38:387–93.
[8] Tsoutsman T, Chung J, Doolan A, et al. Molecular insights from a novel cardiac troponin I mouse model of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 2006;41:623–32. [9] Semsarian C, Ahmad I, Giewat M, et al. The L-type calcium-channel inhibitor diltiazem prevents cardiomyopathy in a mouse model. J Clin Invest 2002;109:1013–20. [10] Schmitt J, Semsarian C, Arad M, et al. Consequences of pressure overload in a murine model of hypertrophic cardiomyopathy. Circulation 2003;108:1133–8.
Letter to the Editor
Abnormal cardiac response to exercise in a murine model of familial hypertrophic cardiomyopathy Lan Nguyen a , Jessica Chung a , Lien Lam a , Tatiana Tsoutsman a,b , Christopher Semsarian a,b,c,⁎ a
Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Australia b Faculty of Medicine, University of Sydney, Australia c Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia Received 8 September 2006; accepted 13 September 2006 Available online 24 October 2006
Abstract Clinical outcome in familial hypertrophic cardiomyopathy (FHC) may be influenced by modifying factors such as exercise. Transgenic mice which overexpress the human disease-causing cTnI gene mutation, Gly203Ser (designated cTnI-G203S), develop all the characteristic phenotypic features of FHC. To study the modifying effect of exercise in early disease, mice underwent swimming exercise at an early age prior to the development of the FHC phenotype. In non-transgenic and cTnI-wt mice, swimming resulted in a significant increase in left ventricular wall thickness and contractility on echocardiography, consistent with a physiological hypertrophic response to exercise. In contrast, cTnI-G203S mice showed no increase in these parameters, indicating an abnormal response to exercise. The lack of a physiological response to exercise may indicate an important novel mechanistic insight into the role of exercise in triggering adverse events in FHC. Crown Copyright © 2006 Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Hypertrophy; Cardiomyopathy; Exercise; Troponin I gene
1. Introduction Familial hypertrophic cardiomyopathy (FHC) is a primary disorder of the myocardium characterised by cardiac hypertrophy in the absence of other loading conditions such as hypertension [1]. Vigorous exercise in FHC has been associated with sudden cardiac death, particularly in the young, and is the basis for the recommendation to FHC patients to avoid competitive sports [2,3]. The mechanistic link between exercise and sudden death in FHC may relate to alterations in Ca2+ homeostasis within cardiomyocytes, changes in energy utilisation, or the histopathological changes, including myocyte disarray and interstitial fibrosis, characteristically seen in
FHC acting as a substrate for ventricular arrhythmias [4,5]. While much is known about how the normal heart responds to exercise, relatively less is known about how this response to exercise is altered in FHC. Since normal Ca2+ handling in cardiac cells is altered during exercise [6], disruptions to components of the sarcomere critical for responding to increases in intracellular Ca2+, e.g. mutations in the cardiac troponin I gene [7], may be important in understanding the association between exercise and sudden death in FHC. This study sought to evaluate the cardiac response to exercise in early (pre-hypertrophic) development of FHC. 2. Materials and methods
⁎ Corresponding author. Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Locked Bag 6, Newtown NSW 2042, Australia. Tel.: +61 2 9565 6195; fax: +61 2 9565 6101. E-mail address: [email protected] (C. Semsarian).
2.1. Mouse model and exercise protocol We have recently described the cTnI-G203S transgenic mouse model, which over-expresses the human disease-
0167-5273/$ - see front matter. Crown Copyright © 2006 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2006.09.001
246
L. Nguyen et al. / International Journal of Cardiology 119 (2007) 245–248
Fig. 1. Changes in lactate level (A) and heart:body weight (B) in exercised (grey bars) versus sedentary (black bars) mice. (⁎p b 0.001 versus sedentary mice; minimum n = 5 per group). Data expressed as mean ± SD. Proposed model for differential response to exercise in normal compared to FHC mice (C).
causing cTnI gene mutation, Gly203Ser, in a cardiac-specific manner [8]. These mice develop all the phenotypic features of FHC by age 20–50 weeks depending on the level of protein expression of the mutant gene [8]. Male mice (n = 10) aged 8– 10 weeks were swum in groups of 5 in a pool where the water was heated to 28–32 °C by a warming tray and maintained at this temperature throughout the swimming period. Mutant cTnI-G203S mice from a low transgene-expression line (later onset of FHC features) were compared to non-transgenic (NTG) mice, and those over-expressing wild-type troponin I (cTnI-wt), which are indistinguishable from NTG mice [8]. Mice were initially swum for 10 min twice daily and the duration increased by 10-min increments daily. Upon reaching 60 min twice daily, this duration was maintained for the remainder of the 4-week exercise period. Mice were monitored daily and weighed weekly. Blood lactate levels were measured to monitor exercise intensity levels. Age and strain matched non-exercised sedentary controls were maintained and handled daily, consistent with the daily handling of the swum mice. All mouse experiments were performed in strict accordance with the Institutional Animal Ethics Committee.
2.2. Echocardiography Mice were evaluated longitudinally using 2D and M-mode mouse echocardiography during the study period. Transthoracic echocardiography was performed using a 7–15 MHz linear array probe and a HDI 5000 ultrasonograph (Acuson) as previously described [9]. 2.3. Histopathology Hearts were excised, washed in 1 × PBS, blot dried, and weighed. Heart weight:body weight ratios were calculated for each mouse. Cardiac tissue was treated for histological examination as described previously [10]. In brief, excised and washed hearts were placed in 10% formaldehyde. Sections were fixed, embedded and tissues stained with either hematoxylin and eosin or Masson's Trichrome. 2.4. Statistical analysis All values are given as mean ± standard deviation. Unpaired t tests were used to determine significant
L. Nguyen et al. / International Journal of Cardiology 119 (2007) 245–248 Table 1 Echocardiographic characteristics of exercised mice Genotype/exercise
HR (bpm)
LVWT (mm)
FS (%)
NTG
502 ± 41 456 ± 25⁎ 531 ± 32 460 ± 30⁎ 512 ± 50 501 ± 24
0.90 ± 0.03 1.08 ± 0.04† 0.90 ± 0.03 1.08 ± 0.02† 0.95 ± 0.04 0.97 ± 0.03
66 ± 2 72 ± 3⁎ 66 ± 2 71 ± 1† 65 ± 3 67 ± 4
cTnI-wt cTnI-G203S
Sedentary Swim Sedentary Swim Sedentary Swim
Data expressed as mean ± SD. HR = heart rate, LVWT = maximal left ventricular wall thickness, FS = fractional shortening, NTG = non-transgenic. ⁎p b 0.001, †p b 0.05 versus sedentary mice of same genotype.
differences between exercised and sedentary mice within genotype groups. For all comparisons, a value of P b 0.05 was considered significant. 3. Results Mice were exercised for a period of 4 weeks by swimming and compared to non-exercised sedentary controls. No sudden deaths occurred during the course of these studies. Swum mice had reduced blood lactate levels (Fig. 1A) compared to non-exercised controls, consistent with chronic exercise. There was no difference in body weight between age-matched sedentary and exercised mice in all study groups over the exercise study period. NTG and cTnI-wt mice had a different cardiac response to exercise compared to the cTnI-G203S FHC mice. NTG and cTnI-wt mice showed a physiological response to exercise characterised by mild cardiac hypertrophy, increased contractile function, and a lower resting heart rate following their 4-week exercise program (Table 1). In contrast, cTnI-G203S mice demonstrated an absence of these physiological responses. Specifically, in NTG and cTnI-wt mice, exercise resulted in a significant increase in heart:body weight ratio (Fig. 1B). Mouse echocardiography showed that LV wall thickness was also increased with exercise, while heart rates in these mice were significantly lower than in non-exercised mice (Table 1). These findings are consistent with a physiological hypertrophic response to exercise. In contrast, cTnI-G203S mice showed no increase in heart:body weight ratio or left ventricular wall thickness on echocardiography, indicating an abnormal response to exercise (Table 1). LV systolic function was increased following the swimming program in NTG and cTnI-wt mice, but unchanged in swum cTnI-G203S mice (Table 1). No changes in histopathology were observed in exercised mice. 4. Discussion FHC mice demonstrate an abnormal response to exercise. NTG and cTnI-wt swum mice developed a normal physiological response to exercise, while cTnI-G203S mice lacked this adaptive response. Such an abnormal response to exercise may be an important substrate for adverse events in FHC associated with competitive exercise.
247
The association of competitive exercise with adverse events, including sudden death, remains a significant clinical problem in FHC. FHC remains the commonest structural cause of sudden cardiac death in those aged under 35 years, including competitive athletes [2,3]. Sudden death occurs either during, or immediately after exercise in up to 70% of patients. While ventricular arrhythmias are the most common cardiac arrhythmia preceding death, the triggering events and the nature of the arrhythmogenic substrate remain unclear. Furthermore, it is well established that in approximately 30% of FHC patients, an abnormal haemodynamic/blood pressure response to exercise is observed, which independently acts as an important prognostic risk indicator. The findings of the current study suggest one possibility for the adverse response to exercise seen in FHC may relate to an abnormal response to exercise whereby the normal physiological adaptations to exercise, including improved cardiac contractility, reduced heart rate, and physiological cardiac hypertrophy, are absent. After a 4-week strenuous swimming program, cTnI-G203S mice did not develop a normal physiological response to exercise as seen in NTG and cTnIwt littermates. Importantly, this failure of physiological adaptation to exercise occurred in cTnI-G203S mice at an age prior to the development of the phenotypic features of FHC, indicating the mutation alone is primarily responsible for the difference in exercise response observed and not secondary changes associated with developed pathological cardiac hypertrophy, diastolic impairment and histopathological changes. Abnormal calcium homeostasis, observed in these FHC mice [8] and in other FHC mouse models [9,10], may be involved in this abnormal response to exercise (unpublished data; Fig. 1C). The clinical heterogeneity seen in FHC, in which affected individuals within the same family, and therefore carrying the same mutation, can have vastly different clinical outcomes, indicates both environmental and genetic modifying influences are important mediators of disease and clinical outcome. Utilising various animal models harbouring different gene mutations, such at the cTnI-G203S model presented here, will allow both increased understanding of disease pathogenesis and to enable modifying factors such as exercise to be evaluated in FHC. Acknowledgements CS is the recipient of a National Health and Medical Research Council Practitioner Fellowship. The research is supported by project grants from the National Heart Foundation and the National Health and Medical Research Council of Australia. References [1] Chung J, Tsoutsman T, Semsarian C. Hypertrophic cardiomyopathy: from gene defect to clinical disease. Cell Res 2003;13:9–20. [2] Maron BJ, Shirani J, Poliac LC, Mathenge R, Roberts WC, Mueller FO. Sudden death in young competitive athletes-clinical, demographic and pathological profiles. JAMA 1996;276:199–204.
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L. Nguyen et al. / International Journal of Cardiology 119 (2007) 245–248
[3] Maron BJ. Sudden death in young athletes. N Engl J Med 2003;349:1064–75. [4] Morita H, Seidman J, Seidman CE. Genetic causes of human heart failure. J Clin Invest 2005;115:518–26. [5] Ashrafian H, Redwood C, Blair E, Watkins H. Hypertrophic cardiomyopathy: a paradigm for myocardial energy depletion. Trends Genet 2003;19:263–8. [6] Lehnart SE, Wehrens XH, Marks AR. Calstabin deficiency, ryanodine receptors, and sudden cardiac death. Biochem Biophys Res Commun 2004;322:1267–79. [7] Doolan A, Tebo M, Ingles J, et al. Cardiac troponin I mutations in Australian families with hypertrophic cardiomyopathy: clinical, genetic and functional consequences. J Mol Cell Cardiol 2005;38:387–93.
[8] Tsoutsman T, Chung J, Doolan A, et al. Molecular insights from a novel cardiac troponin I mouse model of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 2006;41:623–32. [9] Semsarian C, Ahmad I, Giewat M, et al. The L-type calcium-channel inhibitor diltiazem prevents cardiomyopathy in a mouse model. J Clin Invest 2002;109:1013–20. [10] Schmitt J, Semsarian C, Arad M, et al. Consequences of pressure overload in a murine model of hypertrophic cardiomyopathy. Circulation 2003;108:1133–8.