Decreased Expression of Tumor Necrosis Factor-α and Regression of Hypertrophy After Nonsurgical Septal Reduction Therapy for Patients With Hypertrophic Obstructive Cardiomyopathy
Background—Nonsurgical septal reduction therapy (NSRT) is a novel therapeutic strategy for patients with hypertrophic obstructive cardiomyopathy (HOCM). Although the clinical benefits of this technique appear to be clear, the structural and functional changes that lead to improvements in cardiac function are not completely defined. In these studies, we sought to define the effect of NSRT on myocardial function as well as various markers of hypertrophy including the expression of tumor necrosis factor (TNF)-α, a cytokine capable of producing fibrosis, left ventricular hypertrophy (LVH), and cardiomyopathy.
Methods and Results—We performed endomyocardial biopsies of the RV side of the septum and echocardiograms on 15 HOCM patients at baseline and after successful NSRT. Comparative analysis on paired myocardial samples were performed to determine the effects of NSRT on LVH, end-diastolic volume and chamber stiffness, myocyte size, collagen content, and TNF-α levels. At baseline, myocardial TNF-α levels were increased in all patients. After NSRT, myocyte size, collagen content, and TNF-α were significantly decreased. These changes were accompanied by an increase in left ventricular volumes and a reduction in LVH and chamber stiffness.
Conclusions—We suggest that pressure overload in HOCM patients contributes to the development of hypertrophy. These data provide the initial experimental evidence to suggest that TNF-α may play a pathogenetic role in the hypertrophy of pressure overload.
Familial hypertrophic obstructive cardiomyopathy (HOCM) is an autosomal dominant disease resulting from mutations in sarcomeric proteins.1 In 30% of cases, there is dynamic left ventricular (LV) outflow tract obstruction (LVOT), and most therapeutic modalities are aimed primarily at relieving symptoms and LVOT obstruction.2 3 Nonsurgical septal reduction therapy (NSRT) is a novel percutaneous technique pioneered by Sigwart,4 whereby ethanol is injected into one or more septal vessels to induce a controlled infarction. As a result, septal thickness decreases and LVOT widens with elimination of the pressure gradient.5 6 Reduction of the pressure gradient, as induced by NSRT, is associated with increased exercise tolerance,7 8 9 decreased LV hypertrophy (LVH),9 and improved diastolic function.10
Although the clinical benefits of this technique are clear, the molecular mechanisms by which elimination of the pressure gradient improve cardiac performance are not known. However, the reduction in LV mass after NSRT suggests that the myocardial response to pressure load may worsen the degree of hypertrophy.9 Therefore, it seems reasonable to postulate that cardiac growth factors released in response to pressure load may participate in the hypertrophy observed in HOCM patients.
Tumor necrosis factor (TNF)-α is a cytokine induced in the myocardium under volume or pressure overload and is capable of producing LVH and cardiomyopathy.11 More importantly, transgenic mice that overexpress TNF-α in the myocardium develop LVH, dilated cardiomyopathy, and premature death.12 Given the fact that TNF-α may be an important mediator of hypertrophy, we sought to define whether TNF-α was expressed in HOCM myocardium and furthermore to define the effect of pressure gradient elimination by NSRT on myocardial function, cellular markers of hypertrophy, and myocardial TNF-α expression.
The protocol was approved by the institutional review board of the Methodist Hospital and Baylor College of Medicine, and all patients gave written informed consent before participation. The group was composed of 15 consecutive HOCM patients (mean age, 36±15 years; 5 women, 10 men) enrolled for NSRT. Patients had asymmetric septal hypertrophy (septal thickness ≥1.5 cm; septum/posterior wall thickness ≥1.3 cm). All had a dynamic LVOT gradient ≥40 mm Hg at rest or ≥60 mm Hg during dobutamine (5 patients at 5 to 10 μg · kg−1 · min−1). Patients were studied before and 6 weeks after NSRT.
Before NSRT, 2D echocardiography and Doppler echocardiography were performed simultaneously with LV catheterization and endomyocardial biopsy. LV pre-A and end-diastolic pressures were measured (pre-A indicates before the A-wave increase in LV pressure; EDP, immediately before the rise in LV systolic pressure). The pre-A pressure relates well to pulmonary capillary wedge pressure13 and thus was used in lieu of it. LVOT gradient was determined by continuous-wave Doppler.14
Patients were imaged with an Acuson or a Hewlett-Packard ultrasound system equipped with a multifrequency transducer (2.5 and 3.5 MHz) and tissue Doppler program. Conventional parasternal and apical views were obtained. From the apical view, the pulsed Doppler sample volume was placed at the mitral valve annulus and tips, and 5 to 10 cardiac cycles were recorded during normal respiration. All Doppler recordings were made at a sweep speed of 100 mm/s. Pulsed Doppler recordings of LVOT velocity were also applied to measure peak-to-peak A-wave transit time from the mitral valve to the LVOT.15 This interval represents the transit time of the mitral A wave from the mitral annulus to the LVOT and is inversely related to late diastolic LV stiffness.15 Color Doppler was used to assess the severity of mitral regurgitation.16 Tissue Doppler was applied in the 4-chamber view, where a 5-mm sample volume was placed at the lateral border of the mitral annulus and 5 to 10 cycles were recorded.17 Studies were stored on videotape for later analysis.
All measurements were performed by one observer blinded to clinical and biopsy data on an off-line station. LV ejection fraction (LVEF) was calculated by the multiple diameter method.18 LV stroke volume was calculated as the product of mitral annulus area and the velocity-time integral of mitral annular flow.19 To obviate the use of geometric assumptions in the measurement of LV volumes, end-diastolic volume (EDV) was derived as Doppler stroke volume/LVEF. The peak early transmitral velocity (E) and the annular early diastolic velocity (Ea) by tissue Doppler17 were measured as previously described.
Peak LVOT gradient was derived by the modified Bernoulli equation LVOT gradient=4v,2 where v=peak velocity in LVOT by continuous-wave Doppler.14 LV pre-A pressure was estimated by the equation LV pre-A pressure=3.3+[1.1(E/Ea)],17 where Ea is the annular early diastolic velocity at the lateral corner of the mitral annulus (r=0.76, P<0.01). This equation was recently validated in our laboratory and correlates well with catheter measurements of pre-A pressure in HOCM patients.17 Likewise, E/Ea related well to pre-A pressure in this population (r=0.74, P<0.01) and allowed detection of changes in this pressure (r=0.78, P<0.01).
Echocardiographic Evaluation of LV Chamber Stiffness
A wave transit time was recently reported to have a strong correlation with late diastolic LV stiffness.15 Therefore, we examined this time interval. We first evaluated its relation to chamber stiffness by combining simultaneously obtained LV pressures and mitral flow velocity in the catheterization laboratory. Late diastolic LV stiffness was calculated as End-diastolic pressure−Pre-A pressure/left atrial stroke volume. Left atrial stroke volume was in turn computed as the product of ventricular stroke volume19 and atrial filling fraction.20 A good correlation was present between this interval and LV stiffness (r=−0.8, SEE=0.04 mm Hg/mL, P<0.001). The ratio of EDV to pre-A pressure was also used as an index of LV stiffness.
Assessment of LVH
To obviate the use of geometric assumptions, LVH was quantified by the total wall thickness score of Spirito and Maron21 at the mitral valve level and the papillary muscle level in the short-axis views.
All analyses were performed in random order by observers who were blinded to the sample source and date of acquisition. Myocardial biopsies (size, 0.5 to 1 mg; 4 per patient) were obtained with a bioptome under fluoroscopic guidance from the right ventricular (RV) side of the septum before and 6 weeks after NSRT. Although one could be concerned that a biopsy from this side of the septum may not reflect alterations in the LV, Unverferth et al22 have shown in 8 postmortem hearts of HCM patients that the RV septal side has the same amount of fibrous tissue as the LV septal side. Myocardial tissue samples were fixed in 2% paraformaldehyde for 45 minutes immediately on collection. Tissue samples were then dehydrated with graded alcohols followed by clearing in xylene and embedding in paraffin by standard protocols. Five-micron sections were cut, collected on slides, and rehydrated.
Myocardial TNF-α Levels
Myocardial TNF-α content was measured by a semiquantitative analysis of TNF-α–stained myocardial tissue. For TNF-α immunostaining, we used a polyclonal anti–TNF-α antibody (R&D systems) at a 1:300 dilution. Staining was performed with a kit (Vector Laboratories), with a peroxidase-conjugated avidin-biotin system and diaminobenzidine used as substrate. For preliminary experiments, myocardial samples were stained at varying concentrations of anti–TNF-α antibody ranging from a 1:10 to a 1:1000 dilution of antibody. Peak staining always occurred at a 1:300 dilution, and this concentration of antibody was used for all subsequent studies.
Staining for Collagen Content
Total collagen content was determined with the use of picrosirius red (PSR). For collagen I and collagen III content, myocardial tissue sections were stained with a polyclonal rabbit anti-human collagen I antibody and anti-human collagen III antibody (Accurate Chemical & Scientific Corp) at a concentration of 1:20 for each. Standard immunostaining protocols were used with an avidin-biotin complex kit from Vector. Chromagen staining used 3-amino-9-ethyl-carbazo (AEC) as substrate.
Hematoxylin and eosin staining was performed to quantify myocyte size. Myocyte edges were delineated and counted in myocardium before and after NSRT. Myocyte diameter was measured at the level of the nucleus. Thirty myocytes were counted per slide, and the data are expressed as mean±SEM.
Quantitative Analysis of Stained Areas
An investigator blinded to myocardial source performed all analyses. Stained sections were photographed with a Diagnostic Instrument Spot II camera mounted on an Olympus AX-70 microscope. Multiple digital images were taken and stored from each sample stained. Staining was analyzed with Image Pro Plus 4.0 software, with color cube–based selection criteria used for positive staining. Both intensity level (range) and area were analyzed according to the method of Matsuo et al.23 Results in this report are based on area of positive staining within the color spectrum for diaminobenzidine (for TNF-α), AEC (for collagen), or red (for PSR) of all intensities greater than those found in control antibody (IgG)-stained sections without correction for intensity. For TNF-α and collagen, 4 fields were counted, and the results are expressed as mean±SEM. The assay variability (both interobserver and intraobserver) was 10%. However, because variation exists between the intensity of the staining from one experiment to the other, comparisons among groups were only performed within the same experiment. Although absolute values varied from experiment to experiment, the relative amount of immune-positive areas did not change.
A Student’s unpaired t test was used to compare HOCM patients with the normal subjects. Changes in the HOCM group before and after NSRT were evaluated by paired t testing. Regression analysis was performed to evaluate correlations between TNF-α changes and changes in myocardial structure and function. Statistical significance was set at a value of P≤0.05.
Effect of NSRT on Myocardial Function
As previously reported by our group and other investigators, patients had an improvement in their symptomatic status after NSRT (median New York Heart Association class decreasing from III to I at 6 weeks, P<0.01). Importantly, NSRT did not result in changes in ejection fraction (P>0.4) while decreasing the magnitude of hypertrophy (P<0.001), the LVOT gradient (P<0.05), and the LV pre-A pressure (P<0.01). These pressure changes were accompanied by an increase in the EDV (P=0.002), the ratio of EDV to pre-A pressure (P<0.01), and prolongation of the A-wave transient time (P<0.01). In addition, the severity of mitral regurgitation was reduced (P<0.05). The Table⇓ summarizes these data.
Effect of NSRT on Histological Markers of Hypertrophy
Figure 1⇓ shows the change in myocyte size after NSRT (data from two separate experiments). There was a significant reduction in myocyte size after NSRT (average reduction of 61%, P<0.005). Figure 2⇓ shows data from two separate experiments for the change in total collagen, collagen I, and collagen III after NSRT. The average reduction in total collagen was 23% (P<0.005); for collagen I, 33% (P<0.005), and for collagen III, 33% (P<0.005). Figure 3⇓ shows a representative immunostaining for the various collagen isoforms on paired myocardial samples at baseline and after NSRT.
Expression of TNF-α in Myocardium of HOCM Patients
Figure 4A⇓ shows the expression of TNF-α in normal subjects (n=10; mean age, 36 years) and in HOCM patients. Myocardial TNF-α levels at baseline were increased in all patients when compared with control subjects, but a wide variation was present among HOCM patients. A weak and insignificant relation was present between baseline TNF-α level and LVOT gradient (r=0.46, P=0.2). In HOCM patients, baseline myocardial TNF-α levels were compared with levels obtained after NSRT. Figure 4B⇓ shows data from two separate experiments that demonstrate a significant reduction in myocardial TNF-α content after NSRT (average reduction of 36%, P<0.005). Figure 5⇓ shows a representative immunostaining for TNF-α in normal myocardium and in HOCM myocardium at baseline and after NSRT.
Relation Between Changes in TNF-α and Myocardial Structure and LV Function After NSRT
Significant correlations were observed between the improvement in echocardiographic parameters of LV stiffness and the percent reduction in collagen I area (A-wave transit time, r=0.77; EDV/Pre-A, r=0.74; both P<0.01; Figure 6⇓). Nonsignificant trends for correlation were present between the reduction in TNF-α concentration and the decrease in total wall thickness score at the papillary muscle level (r=0.42, R2=0.18; P=0.1) as well as the increase in EDV (r=0.56, R2=0.31; P=0.1).
The findings of this study demonstrate for the first time that HOCM myocardium expresses increased levels of TNF-α. Furthermore, TNF-α levels as well as myocyte size and collagen content are markedly decreased after reduction of the LVOT gradient by NSRT. In addition, LVH and stiffness are also reduced.
TNF-α in HOCM Patients
TNF-α is a cytokine produced in the heart under various forms of stress and capable of inducing hypertrophy and cardiomyopathy in experimental animals.12 24 Furthermore, elevated peripheral TNF-α levels have been found in HOCM patients25 ; therefore, we sought to define whether TNF-α was differentially expressed in HOCM patients with LVOT obstruction and after NSRT. To evaluate this hypothesis further, we performed myocardial TNF-α measurements in paired myocardial samples at baseline and after NSRT in HOCM patients. We determined myocardial but not peripheral TNF-α levels for the following reasons: First, there is no correlation between peripheral and intracardiac TNF-α levels,26 and second, it is possible to create the TNF-α–induced cardiac phenotype with normal concentrations of TNF-α in the circulation.12 We found increased expression of myocardial TNF-α in association with high intraventricular pressure gradients. These levels were dramatically reduced after NSRT. Our results are consistent with the hypothesis that myocardial TNF-α is produced in response to pressure load and that prolonged TNF-α stimulation may contribute to the development of fibrosis27 and hypertrophy.28 29
Effects of NSRT on Myocardial Structure
This study presents novel and important observations showing a reduction in myocyte size and collagen content after elimination of LVOT obstruction by NSRT. These findings are consistent with the concept that hypertrophy in HOCM patients is at least partly reversible and that LVH is intimately linked to fibrosis, as demonstrated in several cardiac disease states including HCM.22 30 Cardiac myocytes are surrounded by an interstitial collagen matrix, which coordinates the transmission of force generated by the myocytes and also determines LV size and stiffness. Collagen I is a relatively stiff material with a high tensile strength that strongly influences the myocardial stress-strain relation. It has a nonlinear elastic behavior, whereby beyond a certain length, stress required for further elongation increases exponentially. Consequently, collagen I contributes to the increase in LV chamber stiffness during the later phases of diastolic filling.30 Increased collagen appears to contribute to the abnormal LV stiffness in patients with aortic stenosis.31 Furthermore, regression of the collagen concentration has been associated with normalization of LV stiffness in spontaneously hypertensive rats.32 To our knowledge, the current investigation is the first to show a reduction in collagen content in HOCM patients after relief of LVOT obstruction. Importantly, this reduction in collagen I was associated with improvement in LV stiffness. Although a significant relation does not prove causality, what is known about the influence of collagen I on myocardial stiffness supports the premise that the observed relation has pathophysiological implications.
Implications for the Role of TNF-α in Other Pressure-Overload Diseases of the Myocardium
This study is also the first to demonstrate in humans that intracardiac TNF-α expression is regulated by changes in pressure load. Although our study examined only patients with HOCM, we suggest that the expression of TNF-α is probably not linked to the genetic defect in HOCM but is rather related to the pressure load imposed by LVOT obstruction. If the genetic defect were the sole factor responsible for the upregulation of this protein, then elimination of the pressure gradient would not have resulted in reduction of intracardiac TNF-α content. We therefore believe that these findings may be applicable to other cardiac disorders characterized by pressure overload.
The number of patients in this study is small, and there was no control group of nonobstructive HCM or HOCM not undergoing NSRT. However, all analyses were performed at baseline and after NSRT; therefore all the observed changes in the examined parameters were attributed to the effects of the procedure. Immunofluorescence techniques are not the best approach to detect TNF-α. Nevertheless, the method we used was highly reproducible and yielded consistent results on two separate experiments. Additionally, given the importance of the observed changes relative to the effects of NSRT, one would have preferred that the biopsy samples had been taken from the LV. However, we were limited by our concern for patient safety. Nevertheless, there is good evidence supporting the use of RV septal biopsies. The study by Unverferth et al22 revealed a similar extent of fibrous tissue in the right and left sides of the septum. Because the contraction load in HOCM is applied to all segments of the ventricle proximal to the obstruction, the changes in the distal half of the septum are most probably reflective of changes in other segments. Regarding the noninvasive assessment of LV stiffness, we applied two different methods that utilize separate echocardiographic measurements to have independent confirmation of the changes in LV stiffness. The findings were similar with both.
This study supports the hypothesis that cardiac TNF-α is expressed in HOCM, at least in part, in response to the pressure overload caused by LVOT obstruction. The relief of obstruction by NSRT was accompanied by reduced expression of this protein along with a reduction in the amount of interstitial collagen and myocyte size. These structural changes were accompanied by an increase in ventricular volume and a decrease in the extent of LVH and chamber stiffness.
This study was supported by grant HL-42550 from the National Heart, Lung, and Blood Institute (Dr Entman), a Smith-Kline Investigator Award (Dr Torre-Amione), a Scientist Development grant from the American Heart Association (0030235N; Dr Nagueh), and The Methodist Hospital Foundation (Dr Torre-Amione). We are indebted to Anna Zamora for secretarial assistance, to Alida J. Evans for technical support, and to Dr Douglas L. Mann for the critical review and scientific support.
Guest Editor for this article was Ulrich Sigwart, MD, Royal Brompton and Harefield NHS Trust, United Kingdom.
- Received October 16, 2000.
- Revision received December 19, 2000.
- Accepted January 3, 2001.
- Copyright © 2001 by American Heart Association
Marian AJ, Roberts R. Recent advances in the molecular genetics of hypertrophic cardiomyopathy. Circulation. 1995;92:1336–1347.
Wigle ED, Rakowski H, Kimball BP, et al. Hypertrophic cardiomyopathy: clinical spectrum and treatment. Circulation. 1995;92:1680–1692.
Knight C, Kurbaan AS, Seggewiss H, et al. Nonsurgical septal reduction therapy for hypertrophic obstructive cardiomyopathy: outcome in the first series of patients. Circulation. 1997;95:2075–2081.
Lakkis NM, Nagueh SF, Kleiman NS, et al. Echocardiography guided ethanol septal reduction for hypertrophic obstructive cardiomyopathy. Circulation. 1998;98:1750–1755.
Nagueh SF, Lakkis NM, Middleton KJ, et al. Changes in left ventricular diastolic function six months after non-surgical septal reduction therapy for hypertrophic obstructive cardiomyopathy. Circulation. 1999;99:344–347.
Kapadia S, Oral H, Lee J, et al. Hemodynamic regulation of tumor necrosis factor-α gene and protein expression in adult feline myocardium. Circ Res. 1997;81:187–195.
Bryant D, Becker L, Richardson J, et al. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-α. Circulation. 1998;97:1375–1381.
Pai RG, Suzuki M, Heywood JT, et al. Mitral A velocity wave transit time to the outflow tract as a measure of left ventricular diastolic stiffness: hemodynamic correlations in patients with coronary artery disease. Circulation. 1994;89:553–557.
Helmcke F, Nanda NC, Hsiung MC, et al. Color Doppler assessment of mitral regurgitation with orthogonal planes. Circulation. 1987;75:175–183.
Nagueh SF, Lakkis NM, Middleton KJ, et al. Doppler estimation of left ventricular filling pressures in patients with hypertrophic cardiomyopathy. Circulation. 1999;99:254–261.
Quinones MA, Waggoner AD, Reduto LA, et al. A new, simplified and accurate method for determining ejection fraction with two-dimensional echocardiography. Circulation. 1981;64:744–753.
Lewis JF, Kuo LC, Nelson JG, et al. Pulsed Doppler echocardiographic determination of stroke volume and cardiac output: clinical validation of two new methods using the apical window. Circulation. 1984;70:425–431.
Bozkurt B, Kribbs S, Clubb M Jr, et al. Pathophysiologically relevant concentrations of tumor necrosis factor-α promote progressive left ventricular dysfunction and remodeling in rats. Circulation. 1998;97:1382–1391.
Matsumori A, Yamada T, Suzuh H, et al. Increased circulating cytokines in patients with myocarditis and cardiomyopathy. Br Heart J. 1994;72:561–566.
Torre-Amione G, Kapadia S, Lee J, et al. Tumor necrosis factor-α and tumor necrosis factor receptors in the failing human heart. Circulation. 1996;93:704–711.
Nakamura K, Mihara K. Inhibitory effects of neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-alpha and angiotensin II. Circulation. 1998;98:794–799.
Yokohama T, Nakano M, Bednarozyk JL, et al. Tumor necrosis factor-alpha provokes a hypertrophic growth response in adult cardiac myocytes. Circulation. 1997;95:1247–1252.
Krayenbuehl HP, Hess OM, Monrad ES, et al. Left ventricular myocardial structure in aortic valve disease before, intermediate, and late after aortic valve replacement. Circulation. 1989;79:744–755.
Brilla CG, Maisch B, Weber KT. Renin-angiotensin system and myocardial collagen matrix remodeling in hypertensive heart disease: in vivo and in vitro studies on collagen matrix regulation. Clin Invest. 1993;71(suppl 5):S35–S41.