© 2014 Wiley Periodicals, Inc.
531
CONGENITAL HEART DISEASE ORIGINAL ARTICLE _____________________________________________________________
Apoptosis of Cardiomyocytes in Children with Right Ventricular Pressure Overload with and without Hypoxemia Flora Hau Fung Tsang, M.B.B.S.,* Pak-Cheong Chow, M.B.B.S.,y Yuen-Yuen Ma, M.Sc.,z Kwan Man, Ph.D.,z Lik-Cheung Cheng, M.B.B.S.,* and Yiu-Fai Cheung, M.D.y *Department of Cardiothoracic Surgery, Queen Mary Hospital, Hong Kong, China; yDepartment of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China; and zDepartment of Surgery, The University of Hong Kong, Hong Kong, China BACKGROUND Cardiomyocyte apoptosis has been implicated in ventricular remodeling and initiation of cardiac failure. We sought to determine the severity of right ventricular (RV) cardiomyocyte apoptosis in cyanotic and acyanotic children with RV pressure overload. Methods: Fourteen patients, seven with tetralogy of Fallot (group I) and seven with pulmonary stenosis and ventricular septal defect (group II), undergoing open-heart surgery were studied. Right ventricular biopsies were examined for cardiomyocyte apoptosis by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. The magnitude of cardiomyocyte apoptosis was related to preoperative oxygen saturation and postoperative inotrope use and hospital stay. Results: Compared with group I patients, group II patients were significantly older at operation (p = 0.002) and had a larger body size (p < 0.01) and higher preoperative oxygen saturation (p = 0.01). The prevalence of cardiomyocyte apoptosis in both group I and II patients as a whole was 0.24 W 0.29% (range, 0% to 1.10%). The prevalence was similar between group I (median 0.30%, range 0% to 1.10%) and group II (median 0.20, range 0% to 0.40%, p = 0.65). The prevalence of cardiomyocyte apoptosis correlated positively with preoperative oxygen saturation on room air (r = S0.69, p < 0.005) and postoperative inotrope score (r = 0.67, p = 0.001). A higher postoperative inotrope score (r = 0.68, p = 0.001) was associated with a significant longer duration of postoperative stay in the hospital. Conclusions: The prevalence of cardiomyocyte apoptosis in the pressure-overloaded right ventricle is related to the severity of hypoxia and may have an impact on postoperative course in terms of early postoperative use of inotropes and duration of hospital stay.
doi: 10.1111/jocs.12337 (J Card Surg 2014;29:531–536)
Loss of cardiomyocytes has been shown to occur in the myocardium secondary to ischemic reperfusion,
Funding: This study was supported by the Children’s Heart Foundation and the Queen Mary Hospital Charitable Trust Training and Research assistance Scheme (TRAS-10-05). Conflict of interest: The authors acknowledge no conflict of interest in the submission. Grant sponsor: Children’s Heart Foundation and the Queen Mary Hospital Charitable Trust Training and Research Assistance Scheme; Grant number: TRAS-10-05 Address for correspondence: Prof. Yiu-Fai Cheung, M.D., Division of Paediatric Cardiology, Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Queen Mary Hospital, 102, Pokfulam Road, Hong Kong, China. Fax: þ86-852-2553-9491; e-mail: [email protected]
infarction, and systemic hypertension.1 Additionally, cardiomyocyte loss has been implicated in ventricular remodeling and initiation of cardiac failure in adults with ischemic and nonischemic heart diseases.2 While mechanisms of death of cardiomyocytes include apoptosis, necrosis, and autophagic cell death,3 the pivotal role of apoptotic cell death in the development of heart failure is increasingly recognized.4 Autopsied specimens obtained from the systemic ventricle of children with a variety of congenital heart diseases have shown evidence of cardiomyocyte apoptosis.5 In particular, specimens obtained from children with volume-overloaded and cyanotic cardiac conditions have been shown to exhibit a greater degree of cardiomyocyte apoptosis compared with those obtained from subjects without heart diseases.6
532
TSANG, ET AL. APOPTOSIS IN PRESSURE-LOADED RV
Right ventricular (RV) dysfunction remains an issue of concern in children with tetralogy of Fallot (TOF) both early and late after surgery. Given the existence of chronic RV pressure overload and hypoxemia in these patients, we hypothesize that apoptosis of cardiomyocytes exists in the right ventricle of TOF patients and has implications for early postoperative outcomes. To test this hypothesis, we determined the severity of cardiomyocyte apoptosis in RV biopsy specimens in children with TOF and acyanotic children with pulmonary stenosis and ventricular septal defect (VSD) undergoing total surgical correction and its relation to postoperative inotrope use and duration of stay in the intensive care unit. MATERIALS AND METHODS Subjects Children undergoing total surgical correction for TOF (group I) and those undergoing surgery for significant pulmonary stenosis in association with VSD (group II) were consecutively recruited over a two-year period. Patients with previous systemic-to-pulmonary arterial shunt insertion were excluded to reduce the confounding influence on ventricular volume load and oxygen saturation. The following demographic and clinical data were collected: preoperative and postoperative echocardiographic and cardiac catheterization findings, age, weight and body surface area at operation, sex, preoperative oxygen saturation on admission for operation, cardiopulmonary bypass and aortic crossclamp duration, postoperative inotropic requirement, postoperative duration of ventilation, and the duration of stay in the intensive care unit and the hospital. The postoperative inotrope score was calculated accordingly to the formula as reported by Wernovsky et al.6 Briefly, the inotrope score was calculated by adding the postoperative maximum doses of dopamine and dobutamine in mg/kg/minute and assigning an arbitrary equivalent value of 10 mg/kg/minute inotrope for each 0.1 mg/kg/minute epinephrine. The study protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster and parents of all patients gave informed consent to the study.
Surgical procedure and myocardial tissue processing The operation was performed under cardiopulmonary bypass at moderate systemic hypothermia (30 to 32 8C). The heart was arrested with intermittent antegrade administration of cold blood cardioplegic solution. The RV outflow tract was opened with inspection of the pulmonary valve and the right ventricle. Infundibular muscle resection, pulmonary valvotomy, and transannular repair of the RV outflow using Gore-Tex (W.L Gore, Flagstaff, AZ, USA) patch were performed as required. Repair of the VSD was performed with a Dacron patch. Right ventricular
J CARD SURG 2014;29:531–536
myocardial specimens were obtained and fixed in 10% buffered formalin and subsequently embedded in paraffin for light microscopic examination. Detection of apoptosis Paraffin sections, each of 4 mm in thickness, of the infundibular muscle myocardial specimen were stained with hematoxylin-eosin staining for histopathological examination. Myocardial apoptosis was examined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). Briefly, the tissue slides were deparaffinized and treated with proteinase K (20 mg/mL) for 30 minutes at 37 8C. After rinsing in phosphate-buffered saline, the slides were incubated with terminal deoxynucleotidyl transferase (TdT) for 30 minutes at 37 8C and then blocked in peroxidase solution for 30 minutes at room temperature. The slides were then developed using diaminobenzidine solution. The number of TUNEL-positive cardiomyocytes was determined in at least 25 optical fields of each specimen. The number of TUNEL-positive cells are calculated using light microscopy (400 magnification) and were expressed as a proportion of TUNEL-positive myocyte nuclei from the total number of myocyte nuclei. Data analysis Data were expressed as median (range). Comparisons of variables between group I and II patients were performed using Mann-Whitney test and Fisher’s exact test where appropriate. Pearson correlation was used to assess for correlations between variables. Results were considered statistically significant with a p-value
531
CONGENITAL HEART DISEASE ORIGINAL ARTICLE _____________________________________________________________
Apoptosis of Cardiomyocytes in Children with Right Ventricular Pressure Overload with and without Hypoxemia Flora Hau Fung Tsang, M.B.B.S.,* Pak-Cheong Chow, M.B.B.S.,y Yuen-Yuen Ma, M.Sc.,z Kwan Man, Ph.D.,z Lik-Cheung Cheng, M.B.B.S.,* and Yiu-Fai Cheung, M.D.y *Department of Cardiothoracic Surgery, Queen Mary Hospital, Hong Kong, China; yDepartment of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China; and zDepartment of Surgery, The University of Hong Kong, Hong Kong, China BACKGROUND Cardiomyocyte apoptosis has been implicated in ventricular remodeling and initiation of cardiac failure. We sought to determine the severity of right ventricular (RV) cardiomyocyte apoptosis in cyanotic and acyanotic children with RV pressure overload. Methods: Fourteen patients, seven with tetralogy of Fallot (group I) and seven with pulmonary stenosis and ventricular septal defect (group II), undergoing open-heart surgery were studied. Right ventricular biopsies were examined for cardiomyocyte apoptosis by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. The magnitude of cardiomyocyte apoptosis was related to preoperative oxygen saturation and postoperative inotrope use and hospital stay. Results: Compared with group I patients, group II patients were significantly older at operation (p = 0.002) and had a larger body size (p < 0.01) and higher preoperative oxygen saturation (p = 0.01). The prevalence of cardiomyocyte apoptosis in both group I and II patients as a whole was 0.24 W 0.29% (range, 0% to 1.10%). The prevalence was similar between group I (median 0.30%, range 0% to 1.10%) and group II (median 0.20, range 0% to 0.40%, p = 0.65). The prevalence of cardiomyocyte apoptosis correlated positively with preoperative oxygen saturation on room air (r = S0.69, p < 0.005) and postoperative inotrope score (r = 0.67, p = 0.001). A higher postoperative inotrope score (r = 0.68, p = 0.001) was associated with a significant longer duration of postoperative stay in the hospital. Conclusions: The prevalence of cardiomyocyte apoptosis in the pressure-overloaded right ventricle is related to the severity of hypoxia and may have an impact on postoperative course in terms of early postoperative use of inotropes and duration of hospital stay.
doi: 10.1111/jocs.12337 (J Card Surg 2014;29:531–536)
Loss of cardiomyocytes has been shown to occur in the myocardium secondary to ischemic reperfusion,
Funding: This study was supported by the Children’s Heart Foundation and the Queen Mary Hospital Charitable Trust Training and Research assistance Scheme (TRAS-10-05). Conflict of interest: The authors acknowledge no conflict of interest in the submission. Grant sponsor: Children’s Heart Foundation and the Queen Mary Hospital Charitable Trust Training and Research Assistance Scheme; Grant number: TRAS-10-05 Address for correspondence: Prof. Yiu-Fai Cheung, M.D., Division of Paediatric Cardiology, Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Queen Mary Hospital, 102, Pokfulam Road, Hong Kong, China. Fax: þ86-852-2553-9491; e-mail: [email protected]
infarction, and systemic hypertension.1 Additionally, cardiomyocyte loss has been implicated in ventricular remodeling and initiation of cardiac failure in adults with ischemic and nonischemic heart diseases.2 While mechanisms of death of cardiomyocytes include apoptosis, necrosis, and autophagic cell death,3 the pivotal role of apoptotic cell death in the development of heart failure is increasingly recognized.4 Autopsied specimens obtained from the systemic ventricle of children with a variety of congenital heart diseases have shown evidence of cardiomyocyte apoptosis.5 In particular, specimens obtained from children with volume-overloaded and cyanotic cardiac conditions have been shown to exhibit a greater degree of cardiomyocyte apoptosis compared with those obtained from subjects without heart diseases.6
532
TSANG, ET AL. APOPTOSIS IN PRESSURE-LOADED RV
Right ventricular (RV) dysfunction remains an issue of concern in children with tetralogy of Fallot (TOF) both early and late after surgery. Given the existence of chronic RV pressure overload and hypoxemia in these patients, we hypothesize that apoptosis of cardiomyocytes exists in the right ventricle of TOF patients and has implications for early postoperative outcomes. To test this hypothesis, we determined the severity of cardiomyocyte apoptosis in RV biopsy specimens in children with TOF and acyanotic children with pulmonary stenosis and ventricular septal defect (VSD) undergoing total surgical correction and its relation to postoperative inotrope use and duration of stay in the intensive care unit. MATERIALS AND METHODS Subjects Children undergoing total surgical correction for TOF (group I) and those undergoing surgery for significant pulmonary stenosis in association with VSD (group II) were consecutively recruited over a two-year period. Patients with previous systemic-to-pulmonary arterial shunt insertion were excluded to reduce the confounding influence on ventricular volume load and oxygen saturation. The following demographic and clinical data were collected: preoperative and postoperative echocardiographic and cardiac catheterization findings, age, weight and body surface area at operation, sex, preoperative oxygen saturation on admission for operation, cardiopulmonary bypass and aortic crossclamp duration, postoperative inotropic requirement, postoperative duration of ventilation, and the duration of stay in the intensive care unit and the hospital. The postoperative inotrope score was calculated accordingly to the formula as reported by Wernovsky et al.6 Briefly, the inotrope score was calculated by adding the postoperative maximum doses of dopamine and dobutamine in mg/kg/minute and assigning an arbitrary equivalent value of 10 mg/kg/minute inotrope for each 0.1 mg/kg/minute epinephrine. The study protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster and parents of all patients gave informed consent to the study.
Surgical procedure and myocardial tissue processing The operation was performed under cardiopulmonary bypass at moderate systemic hypothermia (30 to 32 8C). The heart was arrested with intermittent antegrade administration of cold blood cardioplegic solution. The RV outflow tract was opened with inspection of the pulmonary valve and the right ventricle. Infundibular muscle resection, pulmonary valvotomy, and transannular repair of the RV outflow using Gore-Tex (W.L Gore, Flagstaff, AZ, USA) patch were performed as required. Repair of the VSD was performed with a Dacron patch. Right ventricular
J CARD SURG 2014;29:531–536
myocardial specimens were obtained and fixed in 10% buffered formalin and subsequently embedded in paraffin for light microscopic examination. Detection of apoptosis Paraffin sections, each of 4 mm in thickness, of the infundibular muscle myocardial specimen were stained with hematoxylin-eosin staining for histopathological examination. Myocardial apoptosis was examined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). Briefly, the tissue slides were deparaffinized and treated with proteinase K (20 mg/mL) for 30 minutes at 37 8C. After rinsing in phosphate-buffered saline, the slides were incubated with terminal deoxynucleotidyl transferase (TdT) for 30 minutes at 37 8C and then blocked in peroxidase solution for 30 minutes at room temperature. The slides were then developed using diaminobenzidine solution. The number of TUNEL-positive cardiomyocytes was determined in at least 25 optical fields of each specimen. The number of TUNEL-positive cells are calculated using light microscopy (400 magnification) and were expressed as a proportion of TUNEL-positive myocyte nuclei from the total number of myocyte nuclei. Data analysis Data were expressed as median (range). Comparisons of variables between group I and II patients were performed using Mann-Whitney test and Fisher’s exact test where appropriate. Pearson correlation was used to assess for correlations between variables. Results were considered statistically significant with a p-value
