Physiology & Biochemistry
Exhaustive Exercise-Induced Cardiac Conduction System Injury and Changes of cTnT and Cx43
Authors
Y. Chang, T. Yu, H. Yang, Z. Peng
Affiliation
China Institute of Sport Science (CISS), Sport Health and Rehabilitation Center, Beijing, China
Key words
Abstract
O repeated exhaustive exercise
T
O cardiac conduction system
Up to now, studies of exercise-induced cardiac arrhythmia have focused primarily on the work ing myocardium, with few studies examining to the cardiac conduction system where the rhyth mic and synchronized contraction of the heart is initiated. To explore whether the cardiac conduc tion system is involved in the exercise-induced cardiac injury, we performed histological analy sis of sinoatrial node, atrioventricular node and Purkinje fibers and tested the level of struc tural protein cardiac troponin T and Connexin 43 in Sprague Dawley rats following repeated exhaustive exercise. We found increased col lagen deposition, hyperplasia interstitialis, and enhanced activity of lactate dehydrongenase
O exercise-induced arrhythmia O cardiac troponin T O connexin 43
Introduction accepted after revision
▼
May 12, 2014
The role of regular physical exercise in reducing the risk of cardiovascular diseases has been widely demonstrated [19,25,35], However, unre stricted increase in time and intensity of exercise may not always bring about more benefits to the cardiovascular system. A temporal elevation of cardiac troponin T (cTnT), a serologic biomarker of cardiomyocyte damage, was found in working myocardium both in rats after prolonged exercise [8] and in adolescent and adult subjects follow ing prolonged treadmill exercise [7,33,37], sug gesting cardiac injury following excessive exercise. Recurrent heart injury in individuals performing repeated exhaustive exercise can lead to changes in ventricular functions, cardiac fibrosis and consequently promote arrhythmia [4], Numerous studies have shown that athletes have a higher risk of developing arrhythmia [6,24], Baldesberger et al. compared professional cyclists with golfers who had never performed high-endurance training and found that the inci
Bibliography DOI http://dx.doi.org/
10.1055/S-0034-1384545 Published online: September 25, 2014 IntJ Sports Med 2015; 36: 1 -8 © Georg Thieme Verlag KG S tuttgart • New York ISSN 0172-4622 Correspondence Prof. Yun Chang China Institute of Sport Science (CISS) Sport Health and Rehabilitation Center Road Ti yu guan 11 Beijing 100061 China Tel.: +86/10/87182 526 Fax: +86/10/87182 600 [email protected]
and acid phosphatase in the cardiac conduction system following repeated exhaustive exercise. Mitochondrial alterations, enlarged area of inter calated disc and disappearance of gap junctions were additionally observed through electron transmission microscopy. In addition, significant decreases in cardiac troponin T and Connexin 43 were present in the cardiac conduction sys tem in response to repeated exhaustive exercise. All of these findings demonstrate that repeated exhaustive exercise induces ischemic alterations, damage to cytoskeleton and gap junctions, and tissue fibrosis in the cardiac conduction system in rats. These data may shed a new light on the mechanism of exercised-induced cardiac injury and arrhythmia.
dence of atrial fibrillation (AF) and atrial flutter (AFI) was significantly higher in the cyclist group [3], Furthermore, Karjalainen et al. revealed that vigorous long-term exercise is associated with AF in healthy middle-aged men [18]. All of these data have sparked concern about exerciseinduced heart injury and arrhythmia. It is there fore critical to reveal the mechanism in order to prevent the exercise-induced arrhythmia. Studies have shown that cardiac structural remod eling resulting from the adaption to intense work rates during exercise is one of the mechanisms accounting for the appearance of arrhythmia in long-term endurance athletes [1,9]. Morphologi cal changes include the increased size of cardiac myocyte or hypertrophy, enhanced deposition of extracellular matrix constituents or fibrosis and altered expression level or functions of cardiac structural proteins, such as cTnT. As a cardiac reg ulatory protein for muscle contraction, cTnT is involved in controlling the calcium-mediated interaction between actin and myosin. Its serum level has been found capable of reflecting myocar-
Chang Y et al. Effect of Overloaded Exercise on Cardiac Conduction System... IntJ Sports Med 2015; 3 6 :1 -8
1
2
Physiology & Biochemistry
dial cell damage. In addition, gap junctions function in mediating the cell-to-cell propagation of electrical current flow for the orderly heart contraction. Reduced and heterogeneous connexin (Cx) expression has also been shown to trigger increased fibrosis and produce arrhythmogenic substances, thereby promoting the occurrence of cardiac arrhythmia [17,20]. As is commonly known, the electrical signal that causes the rhythmic and synchronized contractions of the heart is initiated through the cardiac conduction system, which consists of the sinoatrial node (SAN), atrioventricular node (AVN) and Purkinje fibers. Any alteration in the cardiac conducting system may affect the sinus rhythm and consequently be involved in the induction of arrhythmia. However, most of the existing studies on exercise-induced arrhythmia have primarily been conducted on the working myocardium. Alterations in the cardiac conduc tion system caused by long-term vigorous exercise are rarely known. In this article we report that cardiac conduction system underwent ischemic injury and fibrosis following repeated exhaustive exercise in the rat model. In addition to the decreased mRNA and protein level of cTnT and Cx43, remodeling of Cx43 expression was also observed in the cardiac conduction system in response to exhaustive exercise. Our data demonstrate the involvement of the cardiac conduction system in exerciseinduced cardiac injury and indicate the possible role of the car diac conduction system in exercise-induced arrhythmia.
Sample preparation and histological studies Sample preparation
Hearts were removed from the rats in the sedentary group and 4, 12 and 24 h following the last exercise in repeated exhaustive exercise group under anesthesia using 10% chloral hydrate (1 mg/ kg). Hearts were then sliced along the sagittal plane starting from the tip of the apex. Tissues from sinoatrial node and atrioven tricular node were dissected using a Leica laser microdissection system laser microscope cutting machine (Leica LMD system, Germany) according to the instruction printed in the user man ual. The cardiac conduction system was localized under a reflec tive light microscope based on both location and histological characteristics. The sinoatrial node is situated beneath the epicardium at the junction of the superior vena cava and the right atrium. In addition, the sinoatrial node is more loosely arrayed than the cells in the adjoining atrium. In the H&E stain, the AVNislocated in the fibrous ring between the right atrium and ventricle of the heart and stains lighter than the tissue in the adjoining atrium. Purkinje fibers were obtained from the myocardium beneath the inner wall of the right ventricle. Specimens were embedded in paraffin, OTC or Epon 618 for further studies. Histological studies Hematoxylin and eosin (H&E) staining and Masson’s tri chrome staining: Paraffin sections were prepared, sliced (4pm
thick) and processed for H&E and Masson’s thichrome staining. Materials and Methods ▼
Animals
Male Sprague Dawley (SD) rats (8-week-old, 220±8 g) were pur chased from Vital River Laboratories (VRL). The animals were housed and bred in the SPF animal facility of the China Institute of Sport Science with free access to rat chow and water under controlled conditions (room temperature 26±2°C; light/dark cycle 12h/12h; relative humidity 40%-45%). All animal proce dures were in accordance with protocols approved by the national rodent care standards and with the ethical standards of the International Journal of Sport Medicine [15].
Special staining: Basic fuchsine and 2R brilliant green stains were applied to the paraffin sections of the myocardium from the right inner ventricular walls to examine ischemic injury. Enzyme-histochemical staining: Frozen sections of the hearts were prepared in OTCand sliced to 8 pm thick. Acid phosphatase (ACP) and lactate dehydrongenase (LDH) activity was tested in the right ventricular myocardium beneath endocardium using azo-coupling and tetrazole reduction, respectively. Staining results were observed and photographed under a light micro scope (Leica, Germany).
Transmission electron microscopy Repeated exhaustive swimming exercise
Biochemical measurement (ELISA)
Fragments of the inner right ventricle from the rats were imme diately fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide. After being dehydrated in an ethanol gradi ent, the samples were embedded in Epon 618. Ultrathin sections (500A) were cut and stained with uranyl acetate and lead cit rate. Sections were then observed under transmission electron microscope (JOEL JEM1010, Japan). For the quantification, ultrathin sections of the right inner ventricular wall were pre pared, stained and selected according to the principles of cell morphometry and stereology. Several microphotos were taken from each selected ultrathin section, which then were input into the computer to quantify the area of inner membrane and crista of mitochondria (S(Mt)/v)) and the number of mitochondria per area (N(Mt)/a)) using a Quantimet 970 image analyzer (Cam bridge Instruments, Inc., Ann Arbor, Ml, USA).
Whole blood was collected from the rats under anesthesia using 10% chloral hydrate (1 rng/kg) via the abdominal aorta using vacuum blood collection tubes. Sera were separated through centrifugation and stored at -80°C until we performed the ELISA assay to test serum cTnT concentration. The serum cTnT was tested using Duoset ELISA kit (RB, USA) according to the instructions printed in the user manual.
The tissues of heart were embedded in OTC for frozen section preparation (8 pm thickness). Cardiac conduction system was localized and collected by laser microscope cutting machine (LEICA, Germany). The frozen sections were then processed for immunofluorescence staining for Cx43 and cTnT using rabbit
50 adult male SD rats were randomly divided into 2 groups: sed entary control group (C, n=20) and repeated exhaustive exercise group (RE, n=30). Rats in the repeated exercise group were sub jected to swimming with a load on their tails equivalent to 3% of their body weight. The swimming tank was made of PVC, meas ured 1.5 m high and 2 m in diameter, and was filled with water to approximately a depth of 0.6 m high. Water temperature was maintained at 30 ±2 °C. The rats were trained 6 times per week for 2 weeks. During swimming, those rats who sank to the bottom of the tank for 10s and could not return to the water surface were considered to be exhausted [36], Sedentary rats were housed and bred under the same condition but without exercising.
Immunofluorescence
Chang Y et al. Effect of Overloaded Exercise on Cardiac Conduction System... IntJ Sports Med 2015; 3 6 :1 -8
Physiology & Biochemistry
Fig. 1 Serum cTnT level in the rats after performing repeated exhaustive exercise. Serum c ln T level was measured using ELISA kit 4 h, 12h and 24h following the repeated exhaustive exercise and in the sedentary group. Data are mean±SD. *, p
Exhaustive Exercise-Induced Cardiac Conduction System Injury and Changes of cTnT and Cx43
Authors
Y. Chang, T. Yu, H. Yang, Z. Peng
Affiliation
China Institute of Sport Science (CISS), Sport Health and Rehabilitation Center, Beijing, China
Key words
Abstract
O repeated exhaustive exercise
T
O cardiac conduction system
Up to now, studies of exercise-induced cardiac arrhythmia have focused primarily on the work ing myocardium, with few studies examining to the cardiac conduction system where the rhyth mic and synchronized contraction of the heart is initiated. To explore whether the cardiac conduc tion system is involved in the exercise-induced cardiac injury, we performed histological analy sis of sinoatrial node, atrioventricular node and Purkinje fibers and tested the level of struc tural protein cardiac troponin T and Connexin 43 in Sprague Dawley rats following repeated exhaustive exercise. We found increased col lagen deposition, hyperplasia interstitialis, and enhanced activity of lactate dehydrongenase
O exercise-induced arrhythmia O cardiac troponin T O connexin 43
Introduction accepted after revision
▼
May 12, 2014
The role of regular physical exercise in reducing the risk of cardiovascular diseases has been widely demonstrated [19,25,35], However, unre stricted increase in time and intensity of exercise may not always bring about more benefits to the cardiovascular system. A temporal elevation of cardiac troponin T (cTnT), a serologic biomarker of cardiomyocyte damage, was found in working myocardium both in rats after prolonged exercise [8] and in adolescent and adult subjects follow ing prolonged treadmill exercise [7,33,37], sug gesting cardiac injury following excessive exercise. Recurrent heart injury in individuals performing repeated exhaustive exercise can lead to changes in ventricular functions, cardiac fibrosis and consequently promote arrhythmia [4], Numerous studies have shown that athletes have a higher risk of developing arrhythmia [6,24], Baldesberger et al. compared professional cyclists with golfers who had never performed high-endurance training and found that the inci
Bibliography DOI http://dx.doi.org/
10.1055/S-0034-1384545 Published online: September 25, 2014 IntJ Sports Med 2015; 36: 1 -8 © Georg Thieme Verlag KG S tuttgart • New York ISSN 0172-4622 Correspondence Prof. Yun Chang China Institute of Sport Science (CISS) Sport Health and Rehabilitation Center Road Ti yu guan 11 Beijing 100061 China Tel.: +86/10/87182 526 Fax: +86/10/87182 600 [email protected]
and acid phosphatase in the cardiac conduction system following repeated exhaustive exercise. Mitochondrial alterations, enlarged area of inter calated disc and disappearance of gap junctions were additionally observed through electron transmission microscopy. In addition, significant decreases in cardiac troponin T and Connexin 43 were present in the cardiac conduction sys tem in response to repeated exhaustive exercise. All of these findings demonstrate that repeated exhaustive exercise induces ischemic alterations, damage to cytoskeleton and gap junctions, and tissue fibrosis in the cardiac conduction system in rats. These data may shed a new light on the mechanism of exercised-induced cardiac injury and arrhythmia.
dence of atrial fibrillation (AF) and atrial flutter (AFI) was significantly higher in the cyclist group [3], Furthermore, Karjalainen et al. revealed that vigorous long-term exercise is associated with AF in healthy middle-aged men [18]. All of these data have sparked concern about exerciseinduced heart injury and arrhythmia. It is there fore critical to reveal the mechanism in order to prevent the exercise-induced arrhythmia. Studies have shown that cardiac structural remod eling resulting from the adaption to intense work rates during exercise is one of the mechanisms accounting for the appearance of arrhythmia in long-term endurance athletes [1,9]. Morphologi cal changes include the increased size of cardiac myocyte or hypertrophy, enhanced deposition of extracellular matrix constituents or fibrosis and altered expression level or functions of cardiac structural proteins, such as cTnT. As a cardiac reg ulatory protein for muscle contraction, cTnT is involved in controlling the calcium-mediated interaction between actin and myosin. Its serum level has been found capable of reflecting myocar-
Chang Y et al. Effect of Overloaded Exercise on Cardiac Conduction System... IntJ Sports Med 2015; 3 6 :1 -8
1
2
Physiology & Biochemistry
dial cell damage. In addition, gap junctions function in mediating the cell-to-cell propagation of electrical current flow for the orderly heart contraction. Reduced and heterogeneous connexin (Cx) expression has also been shown to trigger increased fibrosis and produce arrhythmogenic substances, thereby promoting the occurrence of cardiac arrhythmia [17,20]. As is commonly known, the electrical signal that causes the rhythmic and synchronized contractions of the heart is initiated through the cardiac conduction system, which consists of the sinoatrial node (SAN), atrioventricular node (AVN) and Purkinje fibers. Any alteration in the cardiac conducting system may affect the sinus rhythm and consequently be involved in the induction of arrhythmia. However, most of the existing studies on exercise-induced arrhythmia have primarily been conducted on the working myocardium. Alterations in the cardiac conduc tion system caused by long-term vigorous exercise are rarely known. In this article we report that cardiac conduction system underwent ischemic injury and fibrosis following repeated exhaustive exercise in the rat model. In addition to the decreased mRNA and protein level of cTnT and Cx43, remodeling of Cx43 expression was also observed in the cardiac conduction system in response to exhaustive exercise. Our data demonstrate the involvement of the cardiac conduction system in exerciseinduced cardiac injury and indicate the possible role of the car diac conduction system in exercise-induced arrhythmia.
Sample preparation and histological studies Sample preparation
Hearts were removed from the rats in the sedentary group and 4, 12 and 24 h following the last exercise in repeated exhaustive exercise group under anesthesia using 10% chloral hydrate (1 mg/ kg). Hearts were then sliced along the sagittal plane starting from the tip of the apex. Tissues from sinoatrial node and atrioven tricular node were dissected using a Leica laser microdissection system laser microscope cutting machine (Leica LMD system, Germany) according to the instruction printed in the user man ual. The cardiac conduction system was localized under a reflec tive light microscope based on both location and histological characteristics. The sinoatrial node is situated beneath the epicardium at the junction of the superior vena cava and the right atrium. In addition, the sinoatrial node is more loosely arrayed than the cells in the adjoining atrium. In the H&E stain, the AVNislocated in the fibrous ring between the right atrium and ventricle of the heart and stains lighter than the tissue in the adjoining atrium. Purkinje fibers were obtained from the myocardium beneath the inner wall of the right ventricle. Specimens were embedded in paraffin, OTC or Epon 618 for further studies. Histological studies Hematoxylin and eosin (H&E) staining and Masson’s tri chrome staining: Paraffin sections were prepared, sliced (4pm
thick) and processed for H&E and Masson’s thichrome staining. Materials and Methods ▼
Animals
Male Sprague Dawley (SD) rats (8-week-old, 220±8 g) were pur chased from Vital River Laboratories (VRL). The animals were housed and bred in the SPF animal facility of the China Institute of Sport Science with free access to rat chow and water under controlled conditions (room temperature 26±2°C; light/dark cycle 12h/12h; relative humidity 40%-45%). All animal proce dures were in accordance with protocols approved by the national rodent care standards and with the ethical standards of the International Journal of Sport Medicine [15].
Special staining: Basic fuchsine and 2R brilliant green stains were applied to the paraffin sections of the myocardium from the right inner ventricular walls to examine ischemic injury. Enzyme-histochemical staining: Frozen sections of the hearts were prepared in OTCand sliced to 8 pm thick. Acid phosphatase (ACP) and lactate dehydrongenase (LDH) activity was tested in the right ventricular myocardium beneath endocardium using azo-coupling and tetrazole reduction, respectively. Staining results were observed and photographed under a light micro scope (Leica, Germany).
Transmission electron microscopy Repeated exhaustive swimming exercise
Biochemical measurement (ELISA)
Fragments of the inner right ventricle from the rats were imme diately fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide. After being dehydrated in an ethanol gradi ent, the samples were embedded in Epon 618. Ultrathin sections (500A) were cut and stained with uranyl acetate and lead cit rate. Sections were then observed under transmission electron microscope (JOEL JEM1010, Japan). For the quantification, ultrathin sections of the right inner ventricular wall were pre pared, stained and selected according to the principles of cell morphometry and stereology. Several microphotos were taken from each selected ultrathin section, which then were input into the computer to quantify the area of inner membrane and crista of mitochondria (S(Mt)/v)) and the number of mitochondria per area (N(Mt)/a)) using a Quantimet 970 image analyzer (Cam bridge Instruments, Inc., Ann Arbor, Ml, USA).
Whole blood was collected from the rats under anesthesia using 10% chloral hydrate (1 rng/kg) via the abdominal aorta using vacuum blood collection tubes. Sera were separated through centrifugation and stored at -80°C until we performed the ELISA assay to test serum cTnT concentration. The serum cTnT was tested using Duoset ELISA kit (RB, USA) according to the instructions printed in the user manual.
The tissues of heart were embedded in OTC for frozen section preparation (8 pm thickness). Cardiac conduction system was localized and collected by laser microscope cutting machine (LEICA, Germany). The frozen sections were then processed for immunofluorescence staining for Cx43 and cTnT using rabbit
50 adult male SD rats were randomly divided into 2 groups: sed entary control group (C, n=20) and repeated exhaustive exercise group (RE, n=30). Rats in the repeated exercise group were sub jected to swimming with a load on their tails equivalent to 3% of their body weight. The swimming tank was made of PVC, meas ured 1.5 m high and 2 m in diameter, and was filled with water to approximately a depth of 0.6 m high. Water temperature was maintained at 30 ±2 °C. The rats were trained 6 times per week for 2 weeks. During swimming, those rats who sank to the bottom of the tank for 10s and could not return to the water surface were considered to be exhausted [36], Sedentary rats were housed and bred under the same condition but without exercising.
Immunofluorescence
Chang Y et al. Effect of Overloaded Exercise on Cardiac Conduction System... IntJ Sports Med 2015; 3 6 :1 -8
Physiology & Biochemistry
Fig. 1 Serum cTnT level in the rats after performing repeated exhaustive exercise. Serum c ln T level was measured using ELISA kit 4 h, 12h and 24h following the repeated exhaustive exercise and in the sedentary group. Data are mean±SD. *, p
