Differential Myolysis of Myocardium and Skeletal Muscle in Hamsters With Dilated Cardiomyopathy

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1 Circ J 2006; 70: Differential Myolysis of Myocardium and Skeletal Muscle in Hamsters With Dilated Cardiomyopathy Beneficial Protective Effect of Diltiazem Yosuke Kato, MS*, **; Mitsunori Iwase, MD*, ; Kenji Takagi, PhD*; Takao Nishizawa, MD ; Hiroaki Kanazawa, MD ; Aya Matsushita, MS*; Hisashi Umeda, MD ; Hideo Izawa, MD ; Akiko Noda, PhD*; Yasuo Koike, MD*; Kohzo Nagata, MD*; Mitsuhiro Yokota, MD Background Although dilated cardiomyopathic hamsters (TO-2) with mutation of the -sarcoglycan gene exhibit histological features of muscular dystrophy, it remains to be elucidated whether both myocardium and skeletal muscle are injured in a similar manner. Methods and Results The progression of myolysis in both myocardium and skeletal muscle were assessed biochemically and pathologically in TO-2 and F1B control hamsters. Left ventricular (LV) function was assessed by echocardiography and cardiac catheterization. Both the plasma concentration of cardiac troponin T and the plasma activity of -hydroxybutyrate dehydrogenase (HBD) peaked at 8 weeks of age, and thereafter reduced greatly in TO-2 hamsters. Activity of creatine kinase (CK) in TO-2 hamsters was significantly greater than in controls throughout the observation period. Pathological findings of both nuclear chain and central nuclei in skeletal muscles were observed in TO-2 hamsters throughout the observation period, suggesting regeneration. LV dysfunction was first evident at 8 weeks of age and deteriorated thereafter in TO-2 hamsters. Treatment of TO-2 hamsters with diltiazem from 5 to 8 weeks of age could avert the LV functional deterioration and the increment in -HBD activity, but CK activity was unchanged. Conclusions Despite myolysis in skeletal muscle occurring consistently throughout the observation period, cardiac myolysis occurred predominantly in the early phase. These initial cardiac events might involve coronary spasm and/or calcium overload in the myocardium. (Circ J 2006; 70: ) Key Words: Dilated cardiomyopathy; Diltiazem; Hamsters; Muscular dystrophy; Myolysis Dilated cardiomyopathy (DCM) is a common cause of heart failure and is multifactorial, resulting from myocarditis, ischemia-induced injury, and mitochondrial or genetic abnormalities. 1 Delta-sarcoglycan (SG) is part of the dystrophin-associated glycoprotein complex, which plays a pivotal role in protecting muscles against mechanical stress by linking the intracellular cytoskeleton and the extracellular matrix. It has been reported that mutation of the -SG gene is the cause of familial and idiopathic DCM and limb-girdle muscular dystrophy (LGMD) 2F. 2,3 Considering that heart failure is a major cause of death in patients with muscular dystrophy, the management of cardiomyopathy associated with muscular dystrophy is very important. TO-2 hamsters with mutation of -SG have been extensively studied for their progressive left ventricular (LV) (Received March 14, 2006; revised manuscript received July 7, 2006; accepted August 22, 2006) *Pathophysiological Laboratory Sciences, Graduate School of Medicine, Nagoya University, Nagoya, **Department of Pathobiological Science and Technology, School of Health Science, Faculty of Medicine, Tottori University, Yonago, Division of Integrated Medicine and Cardiology, Toyota Memorial Hospital, Toyota, Department of Cardiology, Graduate School of Medicine, Nagoya University, Nagoya, School of Nursing, University of Shizuoka, Shizuoka and Department of Genome Science, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan Mailing address: Mitsunori Iwase, MD, Division of Integrated Medicine and Cardiology, Toyota Memorial Hospital, 1-1 Heiwa-cho, Toyota , Japan. mitsunori_iwase@mail.toyota.co.jp dysfunction as a model of human DCM. 4 8 Furthermore, cardiomyopathic hamsters with mutation of -SG, including TO-2 and BIO14.6 hypertrophic cardiomyopathy hamsters, exhibit histological features of muscular dystrophy. 9 Our previous study revealed that the onset of LV dysfunction in TO-2 hamsters occurred around 8 weeks of age, 7 but the relationship between the progression of LV dysfunction and of muscular dystrophy remains obscure. Therefore, we examined whether both the myocardium and skeletal muscle might be injured in a similar manner in TO-2 hamsters. Elucidation of the mechanism for cardiac dysfunction associated with muscular dystrophy might contribute to improved prognosis. Previously, it has been reported that cardiomyopathic hamsters manifest cardiac myolysis from 1 month of age, probably as a result of myocardial ischemia caused by coronary microvascular abnormalities. 10 Furthermore, we have recently reported that coronary microvascular perfusion reveals a greater number of severe vascular irregularities and constrictions in arteriole-sized vessels in TO-2 hamsters as compared with control hamsters. 7 Therefore, we examined the effect of a calcium antagonist, diltiazem (DTZ), on myolysis in the myocardium and skeletal muscle in the early phase of LV dysfunction. Methods Experimental Animals Male TO-2 cardiomyopathic Syrian hamsters (n=52) and

2 1498 KATO Y et al. 80 C until analysis. Plasma concentration of cardiac troponin T (ctnt) was determined by electrochemiluminescence immunoassay (Roche Diagnostics, Nutley, NJ, USA) using human recombinant ctnt. Activity of -hydroxybutyrate dehydrogenase (HBD), which is the relatively cardiac-specific form of lactate dehydrogenase, was determined by the colorimetric method as previously described. 12 Activity of creatine kinase (CK) was determined by enzymatic colorimetric assay (Wako Pure Chemical Industries, Osaka, Japan). Echocardiography Transthoracic echocardiography was performed at 5, 8, 20, and 30 weeks of age under anesthesia, using a 13MHz transducer (Sequia 512, Siemens Medical Solutions, Mountain View, CA, USA) LV end-diastolic (LVDd) and end-systolic (LVDs) dimensions, interventricular septal thickness (IVST), and LV posterior wall thickness (LVPWT) were measured. LV fractional shortening (LVFS) was calculated as: LVFS = [(LVDd LVDs)/LVDd] 100. Mean LV wall thickness (LVWT) was defined as the average of IVST and LVPWT. The ratio of LVDd and mean LVWT (LVDd/mean LVWT) was also calculated for the assessment of LV remodeling. 6 Cardiac Catheterization At 8 and 20 weeks of age a 1.4F micromanometer-tipped catheter (SPR-677, Millar Instruments, Inc, Houston, TX, USA) was advanced into the left ventricle through the right carotid artery for measurement of LV pressure under anesthesia. Electrocardiography, LV pressure, and the first derivative of LV pressure with respect to time (LV dp/dt) were recorded by a multichannel polygraph system (MCS-5000, Fukuda Denshi, Tokyo, Japan). Fig 1. Time course of biochemical data from F1B and TO-2 hamsters. (A) Cardiac troponin T (ctnt), (B) -hydroxybutyrate dehydrogenase (HBD), (C) creatine kinase (CK). Values are means ± SEM. p<0.01 vs age-matched F1B hamsters. ND, not detected. F1B control hamsters (n=32) were obtained from Bio Breeders (Fitchburg, MA, USA) and maintained under constant environmental conditions, with a 12-h light:dark cycle (light on from to h), and free access to food and water. Animal care was in accordance with institutional guidelines, and the experimental protocol was approved by the Committee on Laboratory Animal Utilization of Nagoya University. The investigation conforms to 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). Biochemical Determinations When they were 5, 8, 20, and 30 weeks of age the hamsters were anesthetized by intraperitoneal injection (TO-2: 50 mg/kg; F1B: 65 mg/kg) of sodium pentobarbital (Dainippon Sumitomo Pharma, Osaka, Japan), according to the method of Ryoke et al. 11 Blood samples (2 3 ml) were collected and the plasma was isolated and then stored at Histopathological Examination Both the heart and skeletal muscles were excised, rinsed with saline, and blotted dry. The atria were removed, and both the right and LV free walls plus the septum were separated. These specimens were fixed by immersion in 20% phosphate-buffered formalin (Wako Pure Chemical Industries). The fixed tissue was dehydrated, embedded in paraffin, sectioned at 4 m thickness, and stained with hematoxylin-eosin. Treatment With Calcium Antagonist A calcium antagonist, DTZ (Tanabe Seiyaku, Saitama, Japan), was administered (40 mg/kg per day in drinking water) to the TO-2 hamsters (n=12, selected at random) from 5 to 8 weeks of age. Echocardiography was performed at 5 and 8 weeks of age, and biochemical data (ctnt, - HBD, and CK) were collected at 8 weeks of age. Statistical Analysis Values are expressed as means ± SEM. Differences between 2 groups were analyzed by unpaired Student s t-test. Multiple comparisons were analyzed by 1-way analysis of variance followed by Scheffé s test. A level of p<0.05 was considered statistically significant. Results Biochemical Assessments of Myolysis The time course of the biochemical data for the F1B and TO-2 hamsters is shown in Fig 1. The concentration of

3 Myolysis in DCH Hamsters 1499 Fig 2. Representative left ventricular M- mode echocardiograms in F1B and TO-2 hamsters at 8 and 30 weeks of age. Fig 3. Time course of echocardiographic data from F1B and TO-2 hamsters. (A) Left ventricular end-diastolic dimensions (LVDd), (B) interventricular septal thickness (IVST), (C) the ratio of LVDd and mean left ventricular wall thickness (LVDd/mean LVWT), (D) left ventricular fractional shortening (LVFS). Values are means ± SEM. *p<0.05 and p<0.01 vs age-matched F1B hamsters. ctnt was slightly increased at 5 weeks of age, was maximal at 8 weeks of age, and decreased gradually thereafter. In contrast, ctnt was not detected at all in the F1B hamsters throughout the observation period (Fig 1A). The plasma activity of -HBD in TO-2 hamsters was significantly greater than that in the F1B hamsters at 5 and 8 weeks of age; however, it was similar during the later phases (Fig 1B). CK activity the TO-2 hamsters was significantly greater than that in the F1B hamsters throughout the observation period (Fig 1C). Echocardiography Representative LV M-mode echocardiograms from F1B and TO-2 hamsters at 8 and 30 weeks of age are shown in Fig 2, and the time course of the echocardiographic indices is shown in Fig 3. LVDd (Fig 3A), IVST (Fig 3B), LVDd/mean LVWT ratio (Fig 3C), and LVFS (Fig 3D) were similar in both TO-2 and F1B hamsters at 5 weeks of age. LV dysfunction was first evident in the TO-2 hamsters at 8 weeks of age, when IVST and LVFS were significantly decreased, and LVDd tended to be increased, and the LVDd/mean LVWT ratio was significantly increased, as compared with the F1B hamsters. Thereafter, LV systolic function progressively deteriorated, and the TO-2 hamsters at 30 weeks of age revealed the typical features of DCM characterized by LV dilation, LV wall thinning, and manifest LV systolic dysfunction. In contrast, LV function in the F1B hamsters was unchanged during the observation period.

4 1500 KATO Y et al. Fig 4. Representative left ventricular pressure recordings in F1B and TO-2 hamsters at 20 weeks of age. ECG, electrocardiography; LV dp/dt, first derivative of left ventricular pressure with respect to time; LVP, left ventricular pressure. Table 1 Hemodynamic Data for F1B and TO-2 Hamsters at 8 and 20 Weeks of Age LVESP (mmhg) LVEDP (mmhg) LV dp/dt max (mmhg/s) LV dp/dt min (mmhg/s) 8 weeks 20 weeks 8 weeks 20 weeks 8 weeks 20 weeks 8 weeks 20 weeks F1B 123±4 115±5 0.8± ±1.3 6,746±323 6,154±446 5,704±293 5,048±772 TO-2 106±3* 91±6* 2.1± ±1.8* 5,300±158** 4,408±382* 4,500±150** 3,001±291* Values are means±sem. LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure; LV dp/dt max and LV dp/dt min, maximum and minimum first derivative of left ventricular pressure, respectively. *p<0.05 and **p<0.01 vs F1B hamsters. Cardiac Catheterization Representative LV pressure recordings from the F1B and TO-2 hamsters at 20 weeks of age are shown in Fig4, and the hemodynamic data for F1B and TO-2 hamsters at 8 and 20 weeks of age are summarized in Table 1. LVESP and LV dp/dt max in the TO-2 hamsters were significantly decreased compared with the F1B hamsters at both 8 and 20 weeks of age. LVEDP in the TO-2 tended to be higher than in the F1B hamsters at 8 weeks of age and was significantly elevated at 20 weeks of age compared with the F1B hamsters. LV dp/dt min in the TO-2 hamsters was significantly reduced compared with the F1B hamsters at both 8 and 20 weeks of age. Histopathological Findings Representative light micrographs of the myocardium and skeletal muscle from F1B and TO-2 hamsters are shown in Fig 5. Contraction band necrosis, as well as coagulation necrosis (CN) associated with inflammatory cells in the myocardium, was apparent in the TO-2 hamsters as early as 5 weeks of age (Fig5C). The extent of CN increased in the TO-2 hamsters at 8 weeks of age and gradually increased thereafter. The number of inflammatory cells decreased, and not only prominent fibrosis but also calcified lesions were evident in TO-2 hamsters at 30 weeks of age (Fig5E). On the other hand, nuclear chain as well as central nuclei in skeletal muscles, suggesting regeneration, were detected in TO-2 hamsters throughout the observation period from 5 to 30 weeks of age (Figs 5D,F). No pathological abnormality was detected in the F1B hamsters at any age (Figs 5A,B). Effects of Calcium Antagonist Treatment The biochemical data at 8 weeks of age are summarized in Table 2. The plasma activity of -HBD in the TO-2 hamsters treated with DTZ was significantly less than that in the untreated TO-2 hamsters. However, both the concentration of ctnt and activity of CK were similar in both treated and untreated TO-2 hamsters. Echocardiographic data at 5 and 8 weeks of age are summarized in Table3. At 5 weeks, all echocardiographic indices were similar in both F1B and TO-2 hamsters. LV systolic function deteriorated markedly in the untreated TO-2 hamsters between 5 and 8 weeks of age. At 8 weeks, IVST, LVDd/mean LVWT ratio, and LVFS in TO-2 hamsters treated with DTZ were ameliorated significantly as compared with the untreated TO-2 hamsters, but LVDd was similar in both treated and untreated TO-2 hamsters. Discussion In the present study of TO-2 hamsters with mutation of the -SG gene, we demonstrated that cardiac myolysis and LV dysfunction occur predominantly in the early phase of DCM, whereas myolysis in skeletal muscle is consistent. Furthermore, a calcium antagonist, DTZ, reduced the initial cardiac myolysis and preserved LV systolic function. Differential Myolysis in Myocardium and Skeletal Muscle Mutation of -SG, a component of the dystrophin-associated glycoprotein complex, is the cause of DCM and LGMD 2F, 2,3 and heart failure is a major cause of death in patients with these conditions. In the present study, we found that cardiac myolysis, as assessed by plasma ctnt and -HBD levels, occurred predominantly at a young age (as early as 5 weeks of age in TO-2 hamsters with mutation of -SG). The extent of the cardiac myolysis peaked at

5 Myolysis in DCH Hamsters 1501 Fig 5. Representative light micrographs of myocardium and skeletal muscle from F1B hamsters at 5 weeks of age (A, B) and TO-2 hamsters at 5 and 30 weeks of age (C F). Arrows indicate contraction band necrosis (C, E) or nuclear chain (D, F). Asterisks indicate inflammatory cells (C,E). Table 2 Biochemical Data for F1B and TO-2 Hamsters at 8 Weeks of Age Group ctnt (ng/ml) -HBD (mu h 1 ml 1 ) CK (IU/L) F1B Not detected 67.6± ±19.9 TO-2 untreated 0.66± ±36.0** 949.5±14.4** TO-2 with DTZ 0.64± ±23.4*, 1,009.2±22.4** Values are means±sem. ctnt, cardiac troponin T; -HBD, -hydroxybutyrate dehydrogenase; CK, creatine kinase; DTZ, diltiazem. *p<0.05 and **p<0.01 vs F1B hamsters; p<0.05 vs untreated TO-2 hamsters. Table 3 Echocardiographic Data for F1B and TO-2 Hamsters at 5 and 8 Weeks of Age LVDd (mm) IVST (mm) LVDd/mean LVWT LVFS (%) 5 weeks 8 weeks 5 weeks 8 weeks 5 weeks 8 weeks 5 weeks 8 weeks F1B 3.56± ± ± ± ± ± ± ±0.9 TO-2 untreated 3.70± ± ± ±0.02* 3.98± ±0.17** 52.7± ±2.6** TO-2 with DTZ 3.53± ± ± ± ± ± ± ±1.4 Values are means±sem. LVDd, left ventricular end-diastolic dimension; IVST, interventricular septal thickness; mean LVWT, mean left ventricular wall thickness; LVFS, left ventricular fractional shortening; DTZ, diltiazem *p<0.05 and **p< 0.01 vs F1B hamsters; p< 0.05 and p<0.01 vs untreated TO-2 hamsters. 8 weeks of age, when LV dysfunction became evident for the first time in the development of DCM in the hamsters. Thereafter, the extent of cardiac myolysis decreased, whereas myolysis of skeletal muscle occurred throughout the current observation period from 5 to 30 weeks of age. These findings suggest that cardiac myolysis and LV dysfunction occur predominantly at the early phase in TO-2 cardiomyopathic hamsters, with consistent myolysis of skeletal muscle. To our knowledge, this is the first comprehensive report that correlates the extent of myolysis in the myocardium with that in the skeletal muscle of TO-2 hamsters. Melacini et al similarly reported that cardiac involvement can be detected in the early stage of muscle disease in patients with mutations of SGs. 13 It has been generally thought that the cardiomyocyte is a terminally differentiated cell and is unable to divide and propagate itself after birth. Recently, Beltrami et al reported that there was cell division of cardiomyocytes, even in the human heart, after myocardial infarction; 14 however, the number of dividing cardiomyocytes appear to be insufficient to restore cardiac function. On the other hand, it is well known that injured skeletal muscle can be regenerated by proliferation of undifferentiated satellite cells. In the present study, the pathological findings of both nuclear chain and

6 1502 KATO Y et al. central nuclei in skeletal muscles were observed in TO-2 hamsters throughout the observation period, suggesting regeneration. The differential serial modes of myolysis between myocardium and skeletal muscle could be attributed to the presence or absence of regeneration. Effects of DTZ on Cardiac Myolysis and LV Function It has been suggested that cardiac myolysis in cardiomyopathic hamsters might result from myocardial ischemia caused by coronary microvascular abnormalities. 10 Furthermore, we recently reported that coronary microvascular perfusion revealed a greater number of severe vascular irregularities and constrictions in arteriole-sized vessels in TO-2 hamsters from 4 to 32 weeks of age than in agematched controls. 7 In the present study, we demonstrated that a calcium antagonist, DTZ, could reduce the initial cardiac myolysis, as assessed by -HBD, and preserve LV systolic function. To our knowledge, this is the first report of the beneficial effects of DTZ on cardiac myolysis and LV dysfunction in the early phase of muscle disease in TO- 2 hamsters with mutation of -SG. Our findings also suggest that these initial cardiac events (ie, cardiac myolysis and LV dysfunction) in TO-2 hamsters might involve coronary spasm and/or calcium overload in myocardium. Coral- Vazquez et al indicated that disruption of the SG complex in vascular smooth muscle perturbed vascular function, which initiated cardiomyopathy and exacerbated muscular dystrophy in -SG-deficient mice. 15 On the other hand, it has been reported that disruption of the smooth muscle SG complex is not required for the development of vascular spasm and that vascular spasm arises from a process that is extrinsic to the vascular smooth muscle cells. 16 To elucidate the role of coronary microvascular abnormalities in the development of DCM, further studies including long-term administration of DTZ should be conducted. In this study, the plasma concentration of ctnt was similar in both DTZ treated and untreated TO-2 hamsters. Although ctnt has been used as a sensitive and specific biochemical marker of irreversible myocardial injury, there is evidence that cardiac troponins might be released by other mechanisms, such as reversible myocardial ischemia. 17 Recently, Ammann et al showed that elevated ctnt levels in medical intensive care patients admitted for reasons other than acute coronary syndromes is associated with decreased LV function and higher levels of tumor necrosis factor (TNF)- and interleukin-6, suggesting that TNF- as well as mediators produced by neutrophilic granulocytes may lead to increased permeability of the cardiomyocytic membrane for macromolecules, resulting in leakage of troponin without myocyte necrosis. 18 Taken together, it might be difficult to differentiate the onset of myocardial necrosis and myocardial damage in the absence of necrosis from a subtle elevation in plasma ctnt levels. Effects of DTZ on Myolysis in Skeletal Muscle In this study, DTZ could not prevent the in skeletal myolysis as assessed by CK. Previously, Bhattacharya et al reported that administration of DTZ for 55 days to BIO14.6 hamsters, which are another cardiomyopathic model with mutation of -SG, reduced the concentration of plasma CK. 19 Although the extent and progression of myolysis in TO-2 hamsters might differ from that in BIO14.6 hamsters, it is possible that longer term treatment with DTZ might improve the myolysis of skeletal muscle in the TO-2 hamsters. Conclusions Despite consistent myolysis of skeletal muscle throughout the study period, cardiac myolysis occurred predominantly in the early phase of disease in TO-2 cardiomyopathic hamsters with mutation of the -SG gene. These initial cardiac events might involve coronary spasm and/or myocardial calcium overload. References 1. Kasper EK, Agema WR, Hutchins GM, Deckers JW, Hare JM, Baughman KL. 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