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Relation of serum levels of mast cell tryptase of left ventricular systolic function, left ventricular volume or congestive heart failure
Journal of Cardiac Failure Vol. 10 No. 1 2004
Relation of Serum Levels of Mast Cell Tryptase to Left Ventricular Systolic Function, Left Ventricular Volume or Congestive Heart Failure BHARATHI UPADHYA, MD,1 JIMMY L. KONTOS, MD,2 FARHAD ARDESHIRPOUR, BS,3 JOSEPH PYE, MA,3 WILLIAM S. BOUCHER, BPharm,4 THEOHARIS C. THEOHARIDES, MD, PhD,4,5 GREGORY J. DEHMER, MD,6 AND EFTHYMIOS N. DELIARGYRIS, MD1,7 Winston-Salem, North Carolina; Washington, DC; Chapel Hill, North Carolina; Boston, Massachusetts; Temple, Texas
ABSTRACT Background: Activated mast cells (MC) present in the myocardium of patients with cardiomyopathy may contribute to left ventricular dilatation and systolic dysfunction. We sought to determine whether peripheral levels of tryptase, an MC-specific protease, are related to indices of left ventricular size and function, as well as congestive heart failure (CHF) or coronary artery disease (CAD). Methods and Results: Serum tryptase was measured in 85 patients undergoing cardiac catheterization with left ventriculography and coronary angiography and examined in relation to left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), congestive heart failure (CHF), and angiographically evident CAD. Systemic tryptase levels were lower in patients with increased (⬎90 mL) LVEDV (6.2 [5.3–8.0] mcg/L versus 8.3 [6.6–10.3] mcg/L, P ⫽ .01) and in patients with CHF (6.2 [3.6– 7.3] mcg/L versus 8 [6.2–10] mcg/L, P ⫽ .02) and tended to be lower in patients with depressed (⬍55%) LVEF (6.8 [5.2–9] mcg/L versus 8 [6.3–9.9] mcg/L, P ⫽ NS). Linear regression did not show a significant relationship between tryptase levels with either LVEF or LVEDV. Finally, tryptase levels were consistently elevated in relation to the presence of CAD. Conclusion: Despite increased numbers of MC in the myocardium of patients with cardiomyopathy, systemic levels of MC tryptase appear to be lower in relation to LV systolic dysfunction, LV dilatation, or clinical CHF. In contrast, the presence of angiographically significant CAD is associated with elevated systemic tryptase levels. Key Words: Inflammation, cytokines, cardiomyopathy, coronary artery disease.
An increase in the fibrous components of the extracellular matrix along with destruction of the physiologic myocardial architecture are the pathologic hallmarks of cardiomyopathy.1 Mast cells (MC) are routinely involved in inflammatory and repair processes and, as such, are present in high numbers in the myocardium of patients with cardiomyopathy.2–6 When
activated, MC release a multitude of immunomodulatory and vasoactive mediators including histamine, peptide leukotrienes, and proteases, which can act as mitogens and comitogens for human fibroblasts, resulting in accelerated collagen synthesis and subsequent fibrosis.7–10 Furthermore, MC proteases such as tryptase and chymase can cleave matrix metalloproteinases into active forms, thereby directly contributing to matrix degradation and architectural destruction.11,12 It is, therefore, conceivable that locally activated MC may contribute to the development and progression of cardiomyopathy. Levels of circulating proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α correlate with congestive heart failure (CHF) clinical status and with long-term prognosis.13–17 We recently demonstrated that such cytokine production starts early in the process of systolic dysfunction—even before the onset of clinical CHF.18 Importantly, we have also demonstrated that interleukin-6 levels in CHF patients are similar in the coronary and peripheral circulations, suggesting that peripheral sampling maybe sufficient
From the 1Cardiology Section, Wake Forest University School of Medicine, Winston-Salem, North Carolina; 2Department of Medicine, George Washington University Hospital, Washington, DC; 3University of North Carolina School of Medicine, Chapel Hill, North Carolina; 4Department of Pharmacology and Experimental Therapeutics and 5Internal Medicine, Tufts University School of Medicine, Boston, Massachusetts; 6Scott & White Clinic, Texas A & M School of Medicine, Temple, Texas; and 7Cardiology Department, Athens Medical Center, Athens, Greece. Manuscript received February 6, 2003; revised manuscript received April 15, 2003; revised manuscript accepted June 18, 2003. Reprint requests: Efthymios N. Deliargyris, MD, FACC, FSCAI, Department of Cardiology, Athens Medical Center, 5-7 Distomou St., 15125 Maroussi, Athens, Greece. 1071-9164/$ - see front matter 쑕 2004 Elsevier Inc. All rights reserved. doi:10.1016/S1071-9164(03)00586-4
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when assessing inflammatory cytokines in CHF patients.19 In this study we sought to determine whether serum levels of tryptase, an MC-specific protease, are elevated systemically in relation to indices of impaired cardiac function such as left ventricular (LV) systolic dysfunction, LV dilatation, or clinical CHF.
evaluate possible relationships between tryptase levels and LVEF or LVEDV. Analyses were performed with the SigmaStat statistical software (SPSS 2.0 Inc. Chicago, IL), with P values ⬍.05 deemed significant.
Results Baseline Characteristics
Methods Study Population Tryptase was measured in 85 patients undergoing cardiac catheterization for a variety of clinical indications, including the evaluation of chest pain, abnormal noninvasive testing indicating coronary artery disease (CAD) or the initial evaluation of CHF. There were no specific inclusion criteria, whereas exclusions included cardiac transplantation, ongoing treatment with corticosteroids, and recent (⬍3 months) acute coronary syndrome (unstable angina, non– Q-wave and Q-wave myocardial infarctions). The medical school institutional review board approved the study protocol, and all participants provided informed consent. Definitions Depressed LV function was defined as an LV ejection fraction (LVEF) ⬍55%. Left ventricular dilatation was defined as an LV end-diastolic volume (LVEDV) ⬎90 mL. CAD was defined as at least 1 ⱖ 50% diameter stenosis in any of the 3 major epicardial coronary arteries. CHF was defined as the combination of ⱖNew York Heart Association (NYHA) class II symptoms and the presence of at least 1 of the clinical signs of CHF (pulmonary rales, a third cardiac sound, or lower extremity edema). All patients were clinically compensated at the time of catheterization. Cardiac Catheterization Biplane left ventriculography was performed in a 50⬚ left anterior oblique and 30⬚ right anterior oblique projection. Both LVEDV and LVEF were calculated by the biplane area-length method with Wynne regression formula.20 Coronary angiography was performed in multiple projections for visualization of all coronary artery segments. Percent diameter stenosis was based on the consensus of 2 experienced angiographers. Sampling and Tryptase Measurements Samples were obtained from the femoral artery after sheath insertion and before heparin or contrast administration. Samples were allowed to clot and then centrifuged at room temperature for 5 minutes. The serum was removed and frozen at –80⬚C for analysis at a later date. Tryptase measurements were performed by radioenzymatic assay (UniCap, Pharmacia, Kalamazoo, MI); values are expressed in mcg/L. Statistical Analysis Baseline characteristics are shown as means ⫾ standard deviation for continuous variables and compared with an unpaired t-test, and as percentages for categorical variables and compared with the Fisher’s exact or chi-square test, as appropriate. Tryptase values were not normally distributed and are expressed as a median with 25th to 75th interquartile range and compared with a nonparametric testing (Mann-Whitney rank sum test). Linear regression was used to
Baseline characteristics for all 85 patients are shown in Table 1. Patients were primarily Caucasian males and approximately 2/3 had significant CAD. As expected, in this population, in which most patients underwent cardiac catheterization for the diagnosis of CAD, there was a high prevalence of cardiac risk factors. Mean LVEF and LVEDV for the group as a whole were within the normal range; however, 15% of patients had CHF. Tryptase in Relation to LVEF and LVEDV
We performed separate analyses to determine whether there was an association between systemic tryptase and LVEF and LVEDV. First, we divided the study population according to the presence of LV dysfunction; normal LVEF (ⱖ55%, n ⫽ 56, mean 70.6 ⫾ 8.8%), and depressed LVEF (⬍55%, n ⫽ 29, mean 38.5 ⫾ 12.5%). Baseline characteristics for these groups are shown in Table 2. Patients with depressed LVEF were less frequently hypertensive (P ⬍ .001), less frequently treated with statins (P ⬍ .001), and had higher mean LVEDV (P ⫽ .003). All patients with clinical CHF had a depressed LVEF. Despite large differences in mean LVEF between the 2 groups (70.6 ⫾ 8.8% versus 38.5 ⫾ 12.5%, P ⬍ .0001), median tryptase levels were not significantly different and in fact tended to be lower in patients with depressed LVEF (Fig. 1). In accordance, linear regression analysis of LVEF as a continuous variable again showed no relationship with tryptase levels (r2 ⫽ .005, P ⫽ NS, Fig. 2). Data on LVEDV were available on 75 of the 85 study patients. Systemic tryptase levels were lower among the
Table 1. Baseline Characteristics, All Patients Age (y) Male (%) Caucasian (%) CHF (%) CAD (%) Diabetes (%) Hypertension (%) Hyperlipidemia (%) Smoking (%) LVEF (%) LVEDV (mL) Aspirin (%) ACEI (%) Statins (%)
59.6 ⫾ 11.9 60 (70.6) 69 (81.2) 13 (15.3) 56 (65.9) 24 (28.2) 66 (77.6) 42 (49.4) 55 (64.7) 59.9 ⫾ 18.3 85.3 ⫾ 29.8 59 (69.4) 37 (43.5) 18 (21.2)
CHF, congestive heart failure; CAD, coronary artery disease; LVEF, left ventricular ejection fraction; LVEDV, left ventricular end-diastolic volume; ACEI, angiotensin converting enzyme inhibitors.
Tryptase Levels in CHF
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Table 2. Baseline Characteristics; Classification According to LVEF
Age (y) Caucasian (%) Male (%) CHF (%) CAD (%) Diabetes (%) Hypertension (%) Hyperlipidemia (%) Smoking (%) Aspirin (%) ACEI (%) Statins (%) LVEF (%) LVEDV (ml)
Normal LVEF (⬎55%) (n ⫽ 56)
Depressed LVEF (⬍55%) (n ⫽ 29)
P value
59.1 ⫾ 11.8 46 (82) 38 (68) 0 36 (64) 12 (21) 49 (88) 32 (57) 32 (57) 42 (75) 25 (45) 17 (30) 70.8 ⫾ 8.8 78.5 ⫾ 24.2
60.6 ⫾ 12.2 23 (79) 22 (76) 13 (44.8) 20 (69) 12 (41) 17 (59) 10 (34) 23 (79) 17 (59) 12 (41) 1 (3.5) 38.9 ⫾ 12.5 100.7 ⫾ 35.7
.58 .98 .61 ⬍.001 .85 .09 .006 .08 .07 .19 .95 ⬍.001 ⬍.001 .003
CHF, congestive heart failure; CAD, coronary artery disease; ACEI, angiotensin converting enzyme inhibitors; LVEF, left ventricular ejection fraction; LVEDV, left ventricular end-diastolic volume.
Fig. 2. Single scatter plot depicting tryptase values among the spectrum of left ventricular ejection fraction (LVEF). Linear regression analysis showed no significant association between serum tryptase levels and LVEF.
27 patients with increased LVEDV compared with those with normal LVEDV (Fig. 3). Linear regression analysis did not demonstrate a positive association between tryptase and LVEDV (r2 ⫽ .03, P ⫽ NS, Fig. 4).
we also compared them with the remaining 16 patients with depressed LVEF but no CHF. Once again, CHF patients actually had lower tryptase levels, but, probably because of the smaller sample size for this comparison, this difference was not significant (Fig. 5).
Tryptase and Clinical CHF
Tryptase and CAD
Among the 85 study patients, 13 had ⱖNYHA class II symptoms and all had depressed LVEF. We compared tryptase levels between the 13 CHF patients and the remaining 72 study patients without CHF and found that the presence of CHF was actually associated with lower tryptase levels (Fig. 5). Because all 13 CHF patients had depressed LVEF,
We had previously shown that tryptase levels are elevated in patients with CAD, a large portion (66%) of the present study population.21,22 Consistent with previous reports, CAD patients in the current study had higher tryptase levels compared with patients without CAD (Fig. 6). When we divided the study population based on normal or low LVEF, the
Fig. 1. Box-and-whisker plot depicting tryptase values in patients with normal or depressed left ventricular ejection fraction (LVEF). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
Fig. 3. Box-and-whisker plot depicting tryptase values in patients with normal or increased left ventricular end-diastolic volume (LVEDV). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
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Fig. 4. Single scatter plot depicting tryptase values among the spectrum of left ventricular end-diastolic volume (LVEDV). Linear regression analysis showed no significant association between serum tryptase levels and LVEDV.
prevalence of CAD remained similar in both groups (64% and 69%, respectively) and tryptase levels were again higher among patients with CAD (see Fig. 6).
Fig. 6. Box-and-whisker plot depicting tryptase values based on the presence of coronary artery disease (CAD) in the whole group, and in relation to normal or depressed left ventricular ejection fraction (LVEF). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
Discussion Our data demonstrate that, despite the presence of increased numbers of activated MC in the myocardium of patients with cardiomyopathy, systemic levels of tryptase, a MC-specific mediator, are not elevated and in fact may be reduced in relation to left ventricular systolic dysfunction, left ventricular dilatation, or clinical CHF. Our observations complement the wealth of reports evaluating the systemic profile of inflammatory mediators in CHF patients and suggest that peripheral levels of such mediators are variable and do not necessarily mirror ongoing local myocardial activity.
Fig. 5. Box-and-whisker plot depicting tryptase values based on the presence of congestive heart failure (CHF) in the whole group, and in relation to normal or depressed left ventricular ejection fraction (LVEF). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
A possible explanation for the lack of an association between systemic tryptase levels and indices of left ventricular size and function or clinical CHF may be related to the site of tryptase production. We have previously examined the site of interleukin-6 production in CHF patients with elevated systemic levels and demonstrated similar values observed in both the peripheral and coronary circulations.19 Based on that data, in the current study we limited our sampling to the periphery. It is, however, important to note that there are distinct differences between interleukin-6 and tryptase that may help explain the discrepancy between our previous and current observations. First, interleukin-6 is a ubiquitous cytokine produced by many different cell types, including MC, macrophages, and cardiac myocytes. Tryptase, on the other hand, is a neutral protease exclusive to MC, although not necessarily a measure of MC activity. It is also possible that these 2 mediators exhibit differential release patterns in CHF patients, a phenomenon previously described in other disease states. For instance, interleukin-6 was shown not to necessarily correspond to tryptase release in systemic mastocytosis and to be released separately from tryptase in response to stem cell factor or interleukin-1.23 In addition, tissue hypoperfusion and ischemia are stimuli for interleukin-6 production, whereas it is unknown if they also induce tryptase production.24 It is therefore reasonable to postulate that interleukin-6 and, possibly, other inflammatory mediators, such as tumor necrosis factor-α, are induced in CHF patients because of the low cardiac output state and are produced at multiple hypoperfused sites, thereby leading to elevated peripheral levels. In fact, the prognostic value of these mediators may be related to such a correlation with a low output state. In contrast, tryptase is specific to and only produced by MC and, therefore, may only be elevated in regions rich with activated MC. Such increased MC density
Tryptase Levels in CHF
and activity has indeed been demonstrated within failing hearts compared with control hearts.2–6 Tryptase may therefore be produced, activated, but also consumed at the local level, thereby explaining the lack of systemic tryptase elevations. In fact, an important local MC contribution to fibrosis and architectural myocardial destruction may therefore go undetected through systemic sampling. Unfortunately, the current study design did not include multisite sampling. Timing of collection may also be important because MC release may subside after clinical stabilization. It has been previously reported that acute stress-induced cardiac histamine and interleukin-6 release lasts for only 10 minutes.25 It is possible that hemodynamic compensation before catheterization in our CHF population resulted in reduction in MC mediator release. In accordance with our previous studies, we again demonstrated elevated tryptase levels in patients with angiographically evident CAD.21,22 Similarly, pathologic studies in cardiomyopathy have demonstrated higher MC numbers in failing hearts secondary to an ischemic compared with a nonischemic etiology and more MC in akinetic rather than nonakinetic segments.26 The higher systemic tryptase levels in patients with CAD may be explained by the fact that MC have been consistently demonstrated within atherosclerotic plaques and through direct release into the circulation could contribute to higher circulating tryptase levels.27,28 In conclusion, despite the presence of large numbers of activated MC in the myocardium of patients with cardiomyopathy, systemic tryptase levels—a specific MC protease— are not elevated and may in fact be reduced in relation to a depressed LVEF, increased LVEDV, or clinical CHF. In contrast, the presence of angiographically evident CAD appears to be associated with elevated systemic tryptase levels.
References 1. Mukherjee D, Sen S. Alteration of collagen phenotypes in ischemic cardiomyopathy. J Clin Invest 1991;88:1141–6. 2. Marone G, de Crescenzo G, Adt M, Patella V, Arbustini E, Genovese A. Immunological characterization and functional importance of human heart mast cells. Immunopharmacology 1995;31:1–18. 3. Patella V, Marino I, Arbustini E, Lamparter-Schummert B, Verga L, Adt M, et al. Stem cell factor in mast cells and increased mast cell density in idiopathic and ischemic cardiomyopathy. Circulation 1998; 97:971–8. 4. Patella V, de Crescenzo G, Lamparter-Schummert B, De Rosa G, Adt M, Marone G. Increased cardiac mast cell density and mediator release in patients with dilated cardiomyopathy. Inflamm. Res 1997; 46(Suppl. I):31–2. 5. Patella V. Human heart mast cells: a definitive case of mast cell heterogeneity. Int Arch Allergy Immunol 1995;106:386–93. 6. Marone G. Immunological modulation of human cardiac mast cells. Neurochem Res 1999;24:1195–202. 7. Hatamochi A, Fujiwara K, Ueki H. Effects of histamine on collagen synthesis by cultured fibroblasts derived from guinea pig skin. Arch Dermatol Res 1985;277:60–4.
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8. Baud L, Perez J, Denis M, Ardaillou R. Modulation of fibroblast proliferation by sulfidopeptide leukotrienes: effect of indomethacin. J Immunol 1987;138:1190–5. 9. Ruoss SJ, Hartmann T, Caughey GH. Mast cell tryptase is a mitogen for cultured fibroblasts. J Clin Invest 1991;88:493–9. 10. Theoharides TC. The mast cell: a neuroimmunoendocrine master player. Int J Tissue React 1996;18:1–21. 11. Lees M, Taylor DJ, Woolley DE. Mast cell proteinases activate precursor of collagenase and stromelysin but not of gelatinases A and B. Eur J Biochem 1994;223:171–7. 12. Gruber BL, Marchese MJ, Suzuki K, Okada Y, Nagase H, Ramamurthy NS. Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloprotienase 3 activation. J Clin Invest 1989;84:1657–62. 13. Tsutamoto T, Hisanaga T, Wada A, Maeda K, Ohnishi M, Fukai D, et al. Interleukin-6 spillover in the peripheral circulation increases with the severity of heart failure, and the high plasma level of interleukin6 is an important prognostic predictor in patients with congestive heart failure. J Am Coll Cardiol 1998;31:391–8. 14. Petretta M, Condorelli GL, Spinelli L, Scopacasa F, De Caterina M, Leosco D, et al. Circulating levels of cytokines and their site of production in patients with mild to severe chronic heart failure. Am Heart J 2000;140:E28. 15. MacGowan GA, Mann DL, Kormos RL, Feldman AM, Murali S. Circulating interleukin-6 in severe heart failure. Am J Cardiol 1997; 79:1128–31. 16. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 1990;323:236–41. 17. Roig E, Orus J, Pare C, Azqueta M, Filella X, Perez-Villa F, et al. Serum interleukin-6 in congestive heart failure secondary to idiopathic dilated cardiomyopathy. Am J Cardiol 1998;82:688–90. 18. Raymond RJ, Dehmer GJ, Theoharides TC, Deliargyris EN. Elevated Interleukin-6 patients with asymptomatic mild to moderate left ventricular dysfunction. Am Heart J 2001;141:435–8. 19. Deliargyris EN, Raymond RJ, Theoharides TC, Boucher WS, Tate DA, Dehmer GJ. Sites of interleukin-6 release in patients with acute coronary syndromes and in patients with Congestive cardiac failure. Am J Cardiol 2000;86:913–8. 20. Wynne J, Green LH, Mann T. Estimation of left ventricular volumes in man from biplane cineangiograms filmed in oblique projections. Am J Cardiol 1978;41:726–32. 21. Deliargyris EN, Dehmer GJ, Tate DA, Smith Jr SC, Boucher WS, Theoharides TC. Elevated levels of mast cell mediators in the coronary circulation of patients with coronary artery disease. Atherosclerosis 1999;144(Suppl. I):I-12. 22. Deliargyris EN, Kontos JL, Dehmer GJ, Boucher WS, Theoharide TC. Tryptase, a novel modulator of fibrinogen activity is elevated in patients with coronary artery disease. Blood Coag Fibrinol 1999; 10(Suppl.):S-108. 23. Theoharides TC, Boucher W, Spear K. Serum interleukin reflects disease severity and osteoporosis in mastocytosis patients. Int Arch Allergy Immunol 2002;128:344–50. 24. Yamauchi-Takihara K, Ihara Y, Ogata A, Yoshizaki K, Azuma J, Kishimoto T. Hypoxic stress induces cardiac myocyte-derived interleukin6. Circulation 1995;91:1520–4. 25. Frangogiannis NG, Shimoni S, Chang SM, Ren G, Shan K, Aggeli C, et al. Evidence for an active inflammatory process in the hibernating human myocardium. Am J Pathol 2002;160:1425–33. 26. Kovanen PT. Role of mast cells in atherosclerosis. Chem Immunol 1995;62:132–70. 27. Kelley JL, Chi DS, Abou-Auda W, Smith JK, Krishnaswamy G. The molecular role of mast cells in atherosclerotic cardiovascular disease. Mol Med Today 2000;6:304–8. 28. Huang M, Pang X, Letourneau R, Boucher W, Theoharides TC. Acute stress induces cardiac mast cell activation and histamine release, effects that are increased in Apolipoprotein E knockout mice. Cardiovasc Res 2002;55:150–60.
Relation of Serum Levels of Mast Cell Tryptase to Left Ventricular Systolic Function, Left Ventricular Volume or Congestive Heart Failure BHARATHI UPADHYA, MD,1 JIMMY L. KONTOS, MD,2 FARHAD ARDESHIRPOUR, BS,3 JOSEPH PYE, MA,3 WILLIAM S. BOUCHER, BPharm,4 THEOHARIS C. THEOHARIDES, MD, PhD,4,5 GREGORY J. DEHMER, MD,6 AND EFTHYMIOS N. DELIARGYRIS, MD1,7 Winston-Salem, North Carolina; Washington, DC; Chapel Hill, North Carolina; Boston, Massachusetts; Temple, Texas
ABSTRACT Background: Activated mast cells (MC) present in the myocardium of patients with cardiomyopathy may contribute to left ventricular dilatation and systolic dysfunction. We sought to determine whether peripheral levels of tryptase, an MC-specific protease, are related to indices of left ventricular size and function, as well as congestive heart failure (CHF) or coronary artery disease (CAD). Methods and Results: Serum tryptase was measured in 85 patients undergoing cardiac catheterization with left ventriculography and coronary angiography and examined in relation to left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), congestive heart failure (CHF), and angiographically evident CAD. Systemic tryptase levels were lower in patients with increased (⬎90 mL) LVEDV (6.2 [5.3–8.0] mcg/L versus 8.3 [6.6–10.3] mcg/L, P ⫽ .01) and in patients with CHF (6.2 [3.6– 7.3] mcg/L versus 8 [6.2–10] mcg/L, P ⫽ .02) and tended to be lower in patients with depressed (⬍55%) LVEF (6.8 [5.2–9] mcg/L versus 8 [6.3–9.9] mcg/L, P ⫽ NS). Linear regression did not show a significant relationship between tryptase levels with either LVEF or LVEDV. Finally, tryptase levels were consistently elevated in relation to the presence of CAD. Conclusion: Despite increased numbers of MC in the myocardium of patients with cardiomyopathy, systemic levels of MC tryptase appear to be lower in relation to LV systolic dysfunction, LV dilatation, or clinical CHF. In contrast, the presence of angiographically significant CAD is associated with elevated systemic tryptase levels. Key Words: Inflammation, cytokines, cardiomyopathy, coronary artery disease.
An increase in the fibrous components of the extracellular matrix along with destruction of the physiologic myocardial architecture are the pathologic hallmarks of cardiomyopathy.1 Mast cells (MC) are routinely involved in inflammatory and repair processes and, as such, are present in high numbers in the myocardium of patients with cardiomyopathy.2–6 When
activated, MC release a multitude of immunomodulatory and vasoactive mediators including histamine, peptide leukotrienes, and proteases, which can act as mitogens and comitogens for human fibroblasts, resulting in accelerated collagen synthesis and subsequent fibrosis.7–10 Furthermore, MC proteases such as tryptase and chymase can cleave matrix metalloproteinases into active forms, thereby directly contributing to matrix degradation and architectural destruction.11,12 It is, therefore, conceivable that locally activated MC may contribute to the development and progression of cardiomyopathy. Levels of circulating proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α correlate with congestive heart failure (CHF) clinical status and with long-term prognosis.13–17 We recently demonstrated that such cytokine production starts early in the process of systolic dysfunction—even before the onset of clinical CHF.18 Importantly, we have also demonstrated that interleukin-6 levels in CHF patients are similar in the coronary and peripheral circulations, suggesting that peripheral sampling maybe sufficient
From the 1Cardiology Section, Wake Forest University School of Medicine, Winston-Salem, North Carolina; 2Department of Medicine, George Washington University Hospital, Washington, DC; 3University of North Carolina School of Medicine, Chapel Hill, North Carolina; 4Department of Pharmacology and Experimental Therapeutics and 5Internal Medicine, Tufts University School of Medicine, Boston, Massachusetts; 6Scott & White Clinic, Texas A & M School of Medicine, Temple, Texas; and 7Cardiology Department, Athens Medical Center, Athens, Greece. Manuscript received February 6, 2003; revised manuscript received April 15, 2003; revised manuscript accepted June 18, 2003. Reprint requests: Efthymios N. Deliargyris, MD, FACC, FSCAI, Department of Cardiology, Athens Medical Center, 5-7 Distomou St., 15125 Maroussi, Athens, Greece. 1071-9164/$ - see front matter 쑕 2004 Elsevier Inc. All rights reserved. doi:10.1016/S1071-9164(03)00586-4
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when assessing inflammatory cytokines in CHF patients.19 In this study we sought to determine whether serum levels of tryptase, an MC-specific protease, are elevated systemically in relation to indices of impaired cardiac function such as left ventricular (LV) systolic dysfunction, LV dilatation, or clinical CHF.
evaluate possible relationships between tryptase levels and LVEF or LVEDV. Analyses were performed with the SigmaStat statistical software (SPSS 2.0 Inc. Chicago, IL), with P values ⬍.05 deemed significant.
Results Baseline Characteristics
Methods Study Population Tryptase was measured in 85 patients undergoing cardiac catheterization for a variety of clinical indications, including the evaluation of chest pain, abnormal noninvasive testing indicating coronary artery disease (CAD) or the initial evaluation of CHF. There were no specific inclusion criteria, whereas exclusions included cardiac transplantation, ongoing treatment with corticosteroids, and recent (⬍3 months) acute coronary syndrome (unstable angina, non– Q-wave and Q-wave myocardial infarctions). The medical school institutional review board approved the study protocol, and all participants provided informed consent. Definitions Depressed LV function was defined as an LV ejection fraction (LVEF) ⬍55%. Left ventricular dilatation was defined as an LV end-diastolic volume (LVEDV) ⬎90 mL. CAD was defined as at least 1 ⱖ 50% diameter stenosis in any of the 3 major epicardial coronary arteries. CHF was defined as the combination of ⱖNew York Heart Association (NYHA) class II symptoms and the presence of at least 1 of the clinical signs of CHF (pulmonary rales, a third cardiac sound, or lower extremity edema). All patients were clinically compensated at the time of catheterization. Cardiac Catheterization Biplane left ventriculography was performed in a 50⬚ left anterior oblique and 30⬚ right anterior oblique projection. Both LVEDV and LVEF were calculated by the biplane area-length method with Wynne regression formula.20 Coronary angiography was performed in multiple projections for visualization of all coronary artery segments. Percent diameter stenosis was based on the consensus of 2 experienced angiographers. Sampling and Tryptase Measurements Samples were obtained from the femoral artery after sheath insertion and before heparin or contrast administration. Samples were allowed to clot and then centrifuged at room temperature for 5 minutes. The serum was removed and frozen at –80⬚C for analysis at a later date. Tryptase measurements were performed by radioenzymatic assay (UniCap, Pharmacia, Kalamazoo, MI); values are expressed in mcg/L. Statistical Analysis Baseline characteristics are shown as means ⫾ standard deviation for continuous variables and compared with an unpaired t-test, and as percentages for categorical variables and compared with the Fisher’s exact or chi-square test, as appropriate. Tryptase values were not normally distributed and are expressed as a median with 25th to 75th interquartile range and compared with a nonparametric testing (Mann-Whitney rank sum test). Linear regression was used to
Baseline characteristics for all 85 patients are shown in Table 1. Patients were primarily Caucasian males and approximately 2/3 had significant CAD. As expected, in this population, in which most patients underwent cardiac catheterization for the diagnosis of CAD, there was a high prevalence of cardiac risk factors. Mean LVEF and LVEDV for the group as a whole were within the normal range; however, 15% of patients had CHF. Tryptase in Relation to LVEF and LVEDV
We performed separate analyses to determine whether there was an association between systemic tryptase and LVEF and LVEDV. First, we divided the study population according to the presence of LV dysfunction; normal LVEF (ⱖ55%, n ⫽ 56, mean 70.6 ⫾ 8.8%), and depressed LVEF (⬍55%, n ⫽ 29, mean 38.5 ⫾ 12.5%). Baseline characteristics for these groups are shown in Table 2. Patients with depressed LVEF were less frequently hypertensive (P ⬍ .001), less frequently treated with statins (P ⬍ .001), and had higher mean LVEDV (P ⫽ .003). All patients with clinical CHF had a depressed LVEF. Despite large differences in mean LVEF between the 2 groups (70.6 ⫾ 8.8% versus 38.5 ⫾ 12.5%, P ⬍ .0001), median tryptase levels were not significantly different and in fact tended to be lower in patients with depressed LVEF (Fig. 1). In accordance, linear regression analysis of LVEF as a continuous variable again showed no relationship with tryptase levels (r2 ⫽ .005, P ⫽ NS, Fig. 2). Data on LVEDV were available on 75 of the 85 study patients. Systemic tryptase levels were lower among the
Table 1. Baseline Characteristics, All Patients Age (y) Male (%) Caucasian (%) CHF (%) CAD (%) Diabetes (%) Hypertension (%) Hyperlipidemia (%) Smoking (%) LVEF (%) LVEDV (mL) Aspirin (%) ACEI (%) Statins (%)
59.6 ⫾ 11.9 60 (70.6) 69 (81.2) 13 (15.3) 56 (65.9) 24 (28.2) 66 (77.6) 42 (49.4) 55 (64.7) 59.9 ⫾ 18.3 85.3 ⫾ 29.8 59 (69.4) 37 (43.5) 18 (21.2)
CHF, congestive heart failure; CAD, coronary artery disease; LVEF, left ventricular ejection fraction; LVEDV, left ventricular end-diastolic volume; ACEI, angiotensin converting enzyme inhibitors.
Tryptase Levels in CHF
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Table 2. Baseline Characteristics; Classification According to LVEF
Age (y) Caucasian (%) Male (%) CHF (%) CAD (%) Diabetes (%) Hypertension (%) Hyperlipidemia (%) Smoking (%) Aspirin (%) ACEI (%) Statins (%) LVEF (%) LVEDV (ml)
Normal LVEF (⬎55%) (n ⫽ 56)
Depressed LVEF (⬍55%) (n ⫽ 29)
P value
59.1 ⫾ 11.8 46 (82) 38 (68) 0 36 (64) 12 (21) 49 (88) 32 (57) 32 (57) 42 (75) 25 (45) 17 (30) 70.8 ⫾ 8.8 78.5 ⫾ 24.2
60.6 ⫾ 12.2 23 (79) 22 (76) 13 (44.8) 20 (69) 12 (41) 17 (59) 10 (34) 23 (79) 17 (59) 12 (41) 1 (3.5) 38.9 ⫾ 12.5 100.7 ⫾ 35.7
.58 .98 .61 ⬍.001 .85 .09 .006 .08 .07 .19 .95 ⬍.001 ⬍.001 .003
CHF, congestive heart failure; CAD, coronary artery disease; ACEI, angiotensin converting enzyme inhibitors; LVEF, left ventricular ejection fraction; LVEDV, left ventricular end-diastolic volume.
Fig. 2. Single scatter plot depicting tryptase values among the spectrum of left ventricular ejection fraction (LVEF). Linear regression analysis showed no significant association between serum tryptase levels and LVEF.
27 patients with increased LVEDV compared with those with normal LVEDV (Fig. 3). Linear regression analysis did not demonstrate a positive association between tryptase and LVEDV (r2 ⫽ .03, P ⫽ NS, Fig. 4).
we also compared them with the remaining 16 patients with depressed LVEF but no CHF. Once again, CHF patients actually had lower tryptase levels, but, probably because of the smaller sample size for this comparison, this difference was not significant (Fig. 5).
Tryptase and Clinical CHF
Tryptase and CAD
Among the 85 study patients, 13 had ⱖNYHA class II symptoms and all had depressed LVEF. We compared tryptase levels between the 13 CHF patients and the remaining 72 study patients without CHF and found that the presence of CHF was actually associated with lower tryptase levels (Fig. 5). Because all 13 CHF patients had depressed LVEF,
We had previously shown that tryptase levels are elevated in patients with CAD, a large portion (66%) of the present study population.21,22 Consistent with previous reports, CAD patients in the current study had higher tryptase levels compared with patients without CAD (Fig. 6). When we divided the study population based on normal or low LVEF, the
Fig. 1. Box-and-whisker plot depicting tryptase values in patients with normal or depressed left ventricular ejection fraction (LVEF). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
Fig. 3. Box-and-whisker plot depicting tryptase values in patients with normal or increased left ventricular end-diastolic volume (LVEDV). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
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Journal of Cardiac Failure Vol. 10 No. 1 February 2004
Fig. 4. Single scatter plot depicting tryptase values among the spectrum of left ventricular end-diastolic volume (LVEDV). Linear regression analysis showed no significant association between serum tryptase levels and LVEDV.
prevalence of CAD remained similar in both groups (64% and 69%, respectively) and tryptase levels were again higher among patients with CAD (see Fig. 6).
Fig. 6. Box-and-whisker plot depicting tryptase values based on the presence of coronary artery disease (CAD) in the whole group, and in relation to normal or depressed left ventricular ejection fraction (LVEF). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
Discussion Our data demonstrate that, despite the presence of increased numbers of activated MC in the myocardium of patients with cardiomyopathy, systemic levels of tryptase, a MC-specific mediator, are not elevated and in fact may be reduced in relation to left ventricular systolic dysfunction, left ventricular dilatation, or clinical CHF. Our observations complement the wealth of reports evaluating the systemic profile of inflammatory mediators in CHF patients and suggest that peripheral levels of such mediators are variable and do not necessarily mirror ongoing local myocardial activity.
Fig. 5. Box-and-whisker plot depicting tryptase values based on the presence of congestive heart failure (CHF) in the whole group, and in relation to normal or depressed left ventricular ejection fraction (LVEF). Boxes represent the 25th and 75th percentiles and the horizontal line within the box the median tryptase values (mcg/L). The vertical lines demonstrate the 10th and 90th percentiles, whereas outliers (⬍10% or ⬎90%) are depicted as individual points.
A possible explanation for the lack of an association between systemic tryptase levels and indices of left ventricular size and function or clinical CHF may be related to the site of tryptase production. We have previously examined the site of interleukin-6 production in CHF patients with elevated systemic levels and demonstrated similar values observed in both the peripheral and coronary circulations.19 Based on that data, in the current study we limited our sampling to the periphery. It is, however, important to note that there are distinct differences between interleukin-6 and tryptase that may help explain the discrepancy between our previous and current observations. First, interleukin-6 is a ubiquitous cytokine produced by many different cell types, including MC, macrophages, and cardiac myocytes. Tryptase, on the other hand, is a neutral protease exclusive to MC, although not necessarily a measure of MC activity. It is also possible that these 2 mediators exhibit differential release patterns in CHF patients, a phenomenon previously described in other disease states. For instance, interleukin-6 was shown not to necessarily correspond to tryptase release in systemic mastocytosis and to be released separately from tryptase in response to stem cell factor or interleukin-1.23 In addition, tissue hypoperfusion and ischemia are stimuli for interleukin-6 production, whereas it is unknown if they also induce tryptase production.24 It is therefore reasonable to postulate that interleukin-6 and, possibly, other inflammatory mediators, such as tumor necrosis factor-α, are induced in CHF patients because of the low cardiac output state and are produced at multiple hypoperfused sites, thereby leading to elevated peripheral levels. In fact, the prognostic value of these mediators may be related to such a correlation with a low output state. In contrast, tryptase is specific to and only produced by MC and, therefore, may only be elevated in regions rich with activated MC. Such increased MC density
Tryptase Levels in CHF
and activity has indeed been demonstrated within failing hearts compared with control hearts.2–6 Tryptase may therefore be produced, activated, but also consumed at the local level, thereby explaining the lack of systemic tryptase elevations. In fact, an important local MC contribution to fibrosis and architectural myocardial destruction may therefore go undetected through systemic sampling. Unfortunately, the current study design did not include multisite sampling. Timing of collection may also be important because MC release may subside after clinical stabilization. It has been previously reported that acute stress-induced cardiac histamine and interleukin-6 release lasts for only 10 minutes.25 It is possible that hemodynamic compensation before catheterization in our CHF population resulted in reduction in MC mediator release. In accordance with our previous studies, we again demonstrated elevated tryptase levels in patients with angiographically evident CAD.21,22 Similarly, pathologic studies in cardiomyopathy have demonstrated higher MC numbers in failing hearts secondary to an ischemic compared with a nonischemic etiology and more MC in akinetic rather than nonakinetic segments.26 The higher systemic tryptase levels in patients with CAD may be explained by the fact that MC have been consistently demonstrated within atherosclerotic plaques and through direct release into the circulation could contribute to higher circulating tryptase levels.27,28 In conclusion, despite the presence of large numbers of activated MC in the myocardium of patients with cardiomyopathy, systemic tryptase levels—a specific MC protease— are not elevated and may in fact be reduced in relation to a depressed LVEF, increased LVEDV, or clinical CHF. In contrast, the presence of angiographically evident CAD appears to be associated with elevated systemic tryptase levels.
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