Ann Thorac Surg 2001;71:1154-1159
© 2001 The Society of Thoracic Surgeons
Original article: cardiovascular
Change of c-Myc expression and cardiac hypertrophy in patients with aortic valve replacement
Satoshi Taketani, MDa,
Yoshiki Sawa, MDa,
Hajime Ichikawa, MDa,
Shigeaki Ohtake, MDa,
Motonobu Nishimura, MDa,
Naomasa Kawaguchi, MDb,
Hikaru Matsuda, MDa
a Department of Surgery Course of Interventional Medicine (E1), Osaka University Graduate School of Medicine, Osaka, Japan
b Allied Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan
Accepted for publication November 19, 2000.
Address reprint requests to Dr Sawa, Department of Surgery Course of Interventional Medicine (E1), Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan
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Abstract
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Background. Long-term volume overload to the left ventricle (LV) due to aortic regurgitation (AR) tends to cause severe impairment in LV function that cannot be reversed even with aortic valve replacement (AVR). Recently, we reported that the protooncogene c-myc is related to the onset of the cardiac hypertrophy and LV dysfunction in patients with chronic AR. However, it is still unclear whether c-myc is related to reversibility of the cardiac hypertrophy or LV dysfunction after AVR.
Methods and Results. Twenty patients with isolated chronic AR who underwent AVR were included in this study. LV function was calculated before and after AVR. After AVR, end-systolic volume index (ESVI) and end-diastolic volume index (EDVI) were improved, but not mass index (LVMI). However, normalization of ESVI and EDVI was observed only in 12 and 9 patients, respectively. Preoperatively, c-Myc protein was expressed in the myocardium of 16 out of 20 patients with an average point count of 35 ± 30%. After AVR, c-Myc protein was observed only in 2 patients. Preoperative ejection fraction (EF), ESVI, and postoperative end-systolic stress (ESS)/ESVI had significant correlation to postoperative cell diameter (CD). Percent c-Myc protein expression before the operation was significantly correlated to postoperative CD, ESVI, and ESS/ESVI. Average c-Myc expression was higher in patients who showed normalization of CD and ESS/ESVI after AVR than the patients who did not.
Conclusions. These data suggest that preoperative expression of c-Myc can be indicative of the reversibility of myocardial cellular hypertrophy and LV dysfunction.
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Introduction
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The outcome of aortic valve replacement for aortic regurgitation has been improved because of the development of preoperative management including cardiopulmonary bypass, myocardial protection, and early diagnosis of valvular insufficiency by ultrasound. However, the life expectancy of patients with severe systolic dysfunction due to a chronic volume overload has not been improved, even after successful aortic valve replacement [1–6]. Excess volume overload to the left ventricle in the long term leads to a severe hypertrophy associated with an enlargement of the ventricular cavity. Decompensation of cardiac function begins when the end-systolic volume reaches a certain point [5, 6]. In these patients, the myocardial function after aortic valve replacement is unlikely to be normalized [5, 6]. Microscopic findings show that the myocardial specimens from these patients are associated with an increase in cell diameter and fibrosis [2, 3].
Some protooncogenes, such as c-myc, c-fos, and c-jun, have been reported to be implicated in the pathogenesis of gene-mediated myocardial remodeling in experimental animal hearts [7–9]. Recently, we reported that the protooncogene c-myc is related to the mechanism of extensive cardiac hypertrophy and left ventricular dysfunction in patients with chronic aortic regurgitation [10]. However, the contribution of protooncogenes to the regression of cardiac hypertrophy is still unclear. This study was designed to clarify whether the reversibility of cardiac hypertrophy or the recovery from left ventricular dysfunction after aortic valve replacement is also related to c-myc.
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Material and methods
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Study patients
Twenty consecutive patients with isolated chronic aortic regurgitation who underwent aortic valve replacement in Osaka University Hospital were included in this study. The purpose of this study and the intensive nature of the tests were explained in detail to all the patients. Patients who gave informed consent underwent restudy, and none had complications at the initial or repeat catheterization. There were 16 males and 4 females, with a mean age of 55 ± 12 years old (range 31 to 73 years) at the time of surgery. The underlying pathologies of aortic regurgitation were rheumatic disease in 15 and congenital bicuspid valve in 5. The New York Heart Association class was II in 4 patients, III in 8 patients, and IV in 4 patients at the time of catheterization. The indications for aortic valve replacement were previous history of cardiac failure classed by The New York Heart Association class III in 8 and IV in 12. Aortic regurgitation of more than grade III was assessed by aortic root angiography. The mean interval between the onset of symptoms and the time of surgery was 7 ± 10 years.
Surgical technique
The St. Jude Medical bileaflet valve was used in all patients (25 mm in 10 patients, 27 mm in 10 patients). Normothermic cardiopulmonary bypass and cold blood cardioplegia were employed in every case. The volume of cardioplegic infusion was determined by the method previously reported [11]. The average aortic cross-clamp time was 38 ± 18 minutes. None had major complications after aortic valve replacement. All prosthetic valves were functioning normally without any paravalvular leakage. The simultaneous pressure measurements demonstrated peak systolic gradients across the prosthetic valve of less than 10 mm Hg in all patients.
Catheterization procedure
Patients underwent cardiac catheterization before the operation with mild sedation and local anesthesia. All the medication was withheld for at least 12 to 18 hours before catheterization. Postoperatively, myocardial biopsy was performed using catheters positioned transseptally in the left ventricle via femoral vein puncture. After the measurements of pressure and cardiac output by a dye-dilution method, left ventricular and aortic root angiography were performed to estimate the left ventricular volume and the degree of aortic regurgitation. All patients underwent postoperative catheterization within 24 months (range 12 to 24 months; mean 18 months).
Measurement of left ventricular function
Left ventricular volume was calculated by the area-length method and regression equation [12]. Extrasystolic and postextrasystolic beats were excluded. The earliest satisfactory beats were selected for analysis. The left ventricular wall thickness was measured at the midportion of the anterior wall in the right anterior oblique ventriculogram at end-diastole. The left ventricular mass was determined by the method described by Rackley and associates [13]. Ventricular volume and mass were indexed for body surface area.
End-diastolic volume (EDV) was defined as the largest volume, and end-systolic volume (ESV) as the smallest volume. A left ventricular ejection fraction (EF) was defined as (EDV-ESV)/EDV. A mean velocity of circumferential fiber shortening was defined as (Dd - Ds)/(Dd x ET), where Dd, Ds, and ET were end-diastolic and systolic minor-axis dimension and ejection time, respectively. The minor-axis dimension was calculated as D = 4A/pL, where A and L were area and length, respectively. The ejection time was measured as the time from the initial rise of the aortic pressure tracing to the nadir of the dicrotic notch. End-systolic circumferential wall stress was calculated from Mirsky’s formula [14]. End-systolic thickness was calculated from the end-systolic volume and left ventricular mass, which are assumed to be constant, according to the method of Hugenholtz and associates [15].
Myocardial biopsy
The specimens of left ventricular myocardium were obtained by endomyocardial biopsy at the time of aortic valve replacement and postoperative cardiac catheterization. At the time of aortic valve replacement, under general anesthesia, the patients underwent median sternotomy and cardiopulmonary bypass. After aortic cross-clamp, the aortic root was opened and blood cardioplegia was given antegradely to arrest the heart. The aortic cusps were excised, and three pieces of the myocardium with a diameter of 2 to 3 mm were obtained from the inside of left ventricular with a disposable forceps bioptome (Cordis, Miami, FL). A myocardial biopsy specimen was also obtained at the end of the postoperative cardiac catheterization procedure using a Cordis forceps bioptome introduced into the left ventricular through a Brockenbrough catheter (Bard Inc, Murray Hill, NJ). Two specimens were embedded in Tissue-Tek (Miles Inc, Elkhart, IN). One was placed in liquid nitrogen at -196°C and another was fixed in 10% neutral formaldehyde and embedded in paraffin.
Histological assessment
Eight serial sections were obtained from each specimen embedded in paraffin and stained with hematoxylin and eosin for light microscopic examination. The myocardial cell diameter was determined from 100 measurements of cross-sectioned myocytes at the exact level of the nucleus and calculated with the use of an image analysis system (SPICCA II; Olympus, Tokyo, Japan) by the method of Chalkley and associates [16] and Arai and associates [17]. A cell diameter below 10 µm was considered normal. The percent of interstitial fibrosis (% fibrosis) was determined as an average of the ratios of fibrosis area to the total area from eight sections, and fibrosis area was calculated with the use of an image analysis system (SPICCA II). The fibrosis content was defined as % fibrosis left ventricular mass index.
Immunohistochemical staining of c-Myc
To stain c-Myc protein in the myocardium immunohistochemically, an enzyme-labeled streptavidin technique was performed using the sheep polyclonal antibody against a synthetic peptide sequence that is completely conserved in mouse and human c-Myc protein (OA-11-801; Cambridge Research Biochemicals, Valley Stream, NY). Briefly, eight sections with a thickness of 4 µm were obtained with the use of Cryostat (HM500; Bright, Cambridgeshire, UK) from two frozen specimens, mounted to glass slides, immediately fixed in 95% methanol for 2 minutes, and then air dried. These sections were placed in methanolic hydrogen peroxide (0.3%) for 30 minutes. After rinsing for 5 minutes in phosphate-buffered saline (PBS), the sections were exposed to normal rabbit serum to minimize nonspecific binding. Four sections were incubated with the primary antibody (10 mg/mL), as shown above, overnight at 4°C. The sections were then washed three times for 5 minutes with PBS, incubated with a second antibody, the biotinylated rabbit anti-mouse immunoglobulin (Dako, Tokyo, Japan) for 10 minutes at room temperature, rinsed, and then treated with peroxidase-labeled streptavidin (Dako). The peroxidase activity was visualized in 0.05 M Tris-HCl buffer and 0.01% hydrogen peroxide for 10 minutes at room temperature. Counterstaining with hematoxylin enhanced nuclear details. The other four sections for control were stained without the primary antibody as described above.
To evaluate the degree of c-Myc protein expression in the myocardium, 100 nuclei of the myocardial cells were examined randomly and % c-Myc protein expression was defined as the number of the nuclei in which c-Myc protein was detected.
Normal subject
For a normal reference, values of quantitative angiographic and left ventricular function data from 15 normal subjects (mean age, 44 ± 8 years) who had undergone catheterization for atypical chest pain were obtained.
Statistical analysis
Values of continuous variables are expressed as a mean ± 1 SD. Comparisons of preoperative and postoperative variables in the same patient were made by paired t test. Statistical comparisons were carried out by a one-way analysis of variance (ANOVA) with data from control subjects, and preoperative and postoperative variables.
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Results
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Left ventricular volume and mass
Preoperatively, the average left ventricular end-systolic volume index (LVESVI), left ventricular end-diastolic volume (LVEDVI), and left ventricular mass index (LVMI) were 96 ± 27 mL/m2, 206 ± 37 mL/m2, and 283 ± 44 g/m2, respectively. After aortic valve replacement, all the patients showed significant recovery in LVESVI (53 ± 14 mL/m2, p < 0.01) and LVEDVI (156 ± 32 mL/m2, p < 0.01), but not in LVMI (245 ± 38 g/m2, NS) (Table 1). Normalization of LVESVI, LVEDVI, and LVMI was observed only in 12, 9, and 0 patients, respectively.
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Table 1. Preoperative and Postoperative Measurements of Left Ventricular Function, Left Ventricular Dimensions, and Histology
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Left ventricular function
Preoperatively, the average left ventricular ejection fraction was 49 ± 11%. The ratio between end-systolic stress and left ventricular end-systolic volume index (ESS/ESVI) was 2.4 ± 1.2 Kdyn/cm2/mL/m2. After aortic valve replacement, these patients showed normalization of left ventricular ejection fraction. However, after aortic valve replacement, ESS/ESVI was normalized only in 13 patients. In the other 7 patients, ESS/ESVI was improved but not normalized.
Histological findings
Preoperatively, the average cell diameter and the fibrous content were 24 ± 15 µm and 18.5 ± 7.3 g/m2, respectively. After aortic valve replacement, cell diameter was decreased (15.2 ± 8.5 µm). In 13 patients, cell diameter was decreased below 10 µm, while in the other 7 patients, cell diameter was decreased but remained greater than 10 µm (Fig 1). Not a single patient showed any regression in fibrous content.
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Fig 1. After aortic valve replacement in 13 patients, cell diameter was decreased below 10 µm (open circles), while in the other 7 patients, cell diameter was decreased but remained more than 10 µm (open triangle). The area between the dashed line is the 95% confidence limit of 15 normal subjects.
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The contribution of several indices to the reversibility of cellular hypertrophy
To clarify the elements that might help to predict the reversibility of the cellular hypertrophy, several indices were plotted against the postoperative cellular diameter.
There was a significant correlation between the preoperative ejection fraction and the postoperative cellular diameter (r = -0.83, p < 0.001) (Fig 2A). The preoperative LVESVI also had a significant correlation to the postoperative cell diameter (r = 0.71, p < 0.001) (Fig 2B), while preoperative LVEDVI did not. There was a significant correlation between the postoperative ESS/ESVI and the postoperative cell diameter (r = 0.85, p < 0.001). When the cell diameter becomes normal postoperatively, postoperative ESS/ESVI was also normalized, whereas ESS/ESVI was subnormal when cell diameter remained high (Fig 3).
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Fig 2. There was a significant correlation between the preoperative ejection fraction and the postoperative cellular diameter (A). The preoperative left ventricular end-systolic volume index (ESVI) also had a significant correlation to the postoperative cell diameter (B).
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Fig 3. There was a significant correlation between the postoperative ESS/ESV and the postoperative cell diameter. When the cell diameter becomes normal postoperatively, postoperative ESS/ESV was also normalized. (ESS = end systolic stress; ESV = end systolic volume.)
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Expression of c-Myc protein in the myocardium
Preoperatively, c-Myc protein was expressed in the myocardium of 16 out of 20 patients, with an average point count of 35 ± 30%. After aortic valve replacement, however, c-Myc was observed only in 2 patients (Fig 4).
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Fig 4. c-Myc protein was expressed in the myocardium of 16 out of 20 patients, with an average point count of 35 ± 30%. After aortic valve replacement, however, c-Myc was observed only in 2 patients.
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In the representative case, c-Myc protein was observed in the nuclei of myocardial cells preoperatively (Fig 5A). After aortic valve replacement, c-Myc protein was not detected in the same patient (Fig 5B).
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Fig 5. In immunohistochemical staining (x200), c-Myc protein was observed in the nuclei of myocardial cells preoperatively (A). After aortic valve replacement, c-Myc protein was not detected in the same patient (B).
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Preoperatively expressed c-Myc protein and cardiac hypertrophy
Percent c-Myc protein expression before the operation was significantly correlated to the postoperative cellular diameter (r = -0.83, p < 0.001) (Fig 6A), LVESVI (r = 0.71, p < 0.001) (Fig 6B), and ESS/ESVI (r = -0.84, p < 0.001) (Fig 6C).
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Fig 6. Percent c-Myc protein expression before the operation was significantly correlated to the postoperative cellular diameter (A), LVESVI (B), and ESS/ESVI (C). (ESS = end systolic stress; ESVI = end systolic volume index; LVESVI = left ventricular end-systolic volume index.)
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Comment
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In the present study, chronically volume overloaded hearts due to isolated aortic regurgitation showed expression of c-myc. This expression of c-myc is correlated to the severity of the cardiac hypertrophy. The preoperative expression of c-myc is also related to the reversibility of the cardiac hypertrophy after AVR.
Left ventricular hypertrophy and the effect of aortic valve replacement
A number of studies have been suggesting the importance of aortic valve replacement before the development of a severe left ventricular impairment [1–6]. Taniguchi and associates reported that surgical correction for aortic regurgitation should be considered before the LVESVI exceeds 100 mL/m2 to achieve an improvement in ESS/ESVI. There is also an argument that aortic valve replacement did not improve left ventricular ejection fraction with poor late outcome when the preoperative LVESVI exceeds 100 mL/m2 [5, 6].
Schwarz and associates have shown that the severely depressed left ventricular function in aortic valvular disease is associated with irreversible myocardial ultrastructural changes; hence, left ventricular function also does not fully recover even after aortic valve replacement [18].
In the present study, although most of the patients showed a significant recovery in left ventricular ejection fraction, there was a group of patients in which cellular diameter did not normalize. In these patients, ESS/ESVI was not normalized, either. Therefore, it can be speculated that aortic valve replacement should be planned before the cellular diameter becomes irreversible to achieve better recovery of the ventricular contractility.
Left ventricular hypertrophy and c-Myc expression
Recently, some protooncogenes have been reported to be implicated in the pathogenesis of gene-mediated myocardial remodeling in experimental animal model [7–9]. The mechanism of cardiac hypertrophy is not only c-Myc; many kinds of protooncogenes are induced. In this regard, these other protooncogenes also should be investigated in a future study. In the clinical setting, we have previously reported that c-Myc is related to severe left ventricular hypertrophy and dysfunction in patients with chronic aortic regurgitation. The expression of c-Myc was induced when the myocardial hypertrophy was moderate. Nevertheless, when the myocardial hypertrophy is in the extreme, cardiac myocytes failed to express the c-Myc.
In the present study, when the expression of c-Myc protein was high, ESS/ESVI was normalized after aortic valve replacement with normalization of cellular diameter, while ESS/ESVI remained subnormal in the patients with preoperative lower c-Myc expression. The statistically significant correlation of percent c-Myc expression to the postoperative cellular diameter, LVESVI, and ESS/ESVI suggests the contribution of c-Myc to both morphological and functional change in the myocardial cells. Postoperatively, c-Myc was undetectable when the left ventricular contractility normalized, while c-Myc was detectable in some patients whose left ventricular contractility remained subnormal. These data suggest that the expression of c-Myc begins to increase at the initiation period of ventricular hypertrophy and ends up with a failure to express c-Myc when the damage is prolonged. Our speculation is that the preoperative expression of c-Myc can be indicative of the reversibility of the myocardial cellular hypertrophy.
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References
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Abstract
Introduction
