Increased Myocardial Gene Expression of Tumor Necrosis Factor-α and Nitric Oxide Synthase-2
A Potential Mechanism for Depressed Myocardial Function in Hibernating Myocardium in Humans
Background— Whether cardioinhibitory cytokines are elevated in regions of hibernating myocardium and account in part for the depression in resting function is currently not known.
Methods and Results— Thirteen patients with stable ischemic ventricular dysfunction scheduled for bypass surgery underwent preoperative dobutamine echocardiography (DE) and intraoperative myocardial biopsies. The numbers of copies of mRNA for the negatively inotropic cytokines tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (NOS2) were quantified by reverse transcription–polymerase chain reaction. In normal segments, myocardial TNF-α was barely detectable (1.2±0.4 copies per 106 copies of β-actin). A 13.7-fold increase in myocardial TNF-α was observed in dysfunctional segments with a biphasic response to DE (contractile reserve and ischemia) and was highest (45.5-fold) in segments with ischemia and without contractile reserve (P<0.001). A similar graded increase was seen for NOS2. Cytokine results were also similar if analysis was performed using recovery of function at 3 months as the index of viability. The change in serum TNF-α and nitrite levels from baseline to 3 months after surgery correlated inversely with both the change in ejection fraction and the number of DE viable segments (r=−0.92 to −0.93; P<0.001).
Conclusions— TNF-α and NOS2 gene expression is regionally upregulated in hibernating myocardium to a level intermediate between that of normal regions and ischemic regions without contractile reserve. This, along with a decline in serum cytokine levels after revascularization proportional to the extent of myocardial viability, suggests a contributing role for cardioinhibitory cytokines in the observed depression of function seen in hibernating myocardium.
Received December 10, 2001; revision received January 18, 2002; accepted February 5, 2002.
The exact mechanisms for depression of contractility in hibernating myocardium (HM) are not fully known. The severity of chronic hypoperfusion, 1 myocardial structural alterations, 2 and regional changes in adrenoreceptor density,3 however, plays a role. Cytokines are upregulated in several clinical settings of contractile dysfunction, including heart failure, sepsis, myocarditis, and ischemia.4 Among these, tumor necrosis factor-α (TNF-α) and nitric oxide (NO) have been shown to depress myocardial contractility.5 We therefore postulated that these cytokines would be regionally upregulated in areas of HM compared with normal myocardium. Myocardial TNF-α and NO synthase (NOS2) mRNA were quantified in normal and dysfunctional ischemic myocardium with and without evidence of contractile reserve by dobutamine echocardiography (DE) in patients undergoing coronary artery bypass surgery. We also explored the relation between the extent of myocardial viability and the change in serum levels of cytokines after revascularization.
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Patients with chronic stable ischemic left ventricular dysfunction in the distribution of ≥1 significant coronary artery stenoses (≥70%) who were already scheduled for bypass surgery were enrolled. Study results did not change their management. Patients were excluded if they had a history of unstable angina or infarction in the previous 3 months, decompensated heart failure, ventricular tachycardia, and significant valvular or systemic disease. The Institutional Review Board of Baylor College of Medicine approved the protocol, and all patients gave written informed consent before enrollment.
To assess myocardial viability, DE was performed within 1 to 3 days before surgery as previously described.2 Myocardial thickening fraction2 and ejection fraction (EF) were quantified6 as previously described. Recovery of regional function was defined as an improvement of ≥2 grades in wall motion.2 The responses of dysfunctional segments to DE were classified as biphasic, sustained improvement, worsening, and no change. A biphasic response (presence of contractile reserve and ischemia) has the highest predictive value for recovery of function (72%) and is associated with minimal fibrosis, whereas the worsening response connotes lack of contractile reserve, the presence of severe ischemia with an intermediate likelihood for recovery of 30%, and more fibrosis.2,7⇓ Three months later, DE was repeated. All studies were randomized and interpreted without knowledge of clinical, histopathological, or cytokine data.
Transmural Biopsies and Cytokine Quantification
Transmural myocardial biopsies from the anterior, inferior, or lateral walls were obtained with a 20-mm, 14-gauge Tru-cut biopsy needle at the time of bypass surgery, before cardioplegia, guided by transesophageal echocardiography. Two biopsies were acquired per patient, 1 from a dysfunctional segment and the other from a normal segment for use as control when feasible. Segments with scar and a very low likelihood of viability (thickness ≤0.6 cm) were not biopsied.
The numbers of mRNA copies for TNF-α and NOS2 were determined by use of real-time quantitative reverse transcription–polymerase chain reaction LightCycler technology8 and were indexed to per million copies of β-actin (housekeeping gene). This indexing was performed to normalize for potential differences in the amount of fibrosis in different regions. Results were similar if analysis was done by indexing to weight of the myocardial samples. Serum was assayed for levels of TNF-α by ELISA and nitrite (breakdown product of NO) by the Griess reaction, as previously described by us.9 Serum samples were collected before the preoperative DE was started and at the time of the follow-up visit.
Data are expressed as mean±SEM. One-way ANOVA was used to test for differences between group means. Paired t test was used to test for differences between paired data before and after surgery. Linear regression or Pearson correlation was used to correlate changes in EF or cytokines and the extent of viability. Significance was set at a value of P<0.05.
The population consisted of 13 patients (10 men) with a mean age of 62±2 years and EF of 31±3%. The patients’ NYHA functional class averaged 2.7±0.2. Eight patients had stable angina, 7 had a remote history of infarction, 6 were diabetic, and 10 had hypertension. Nine patients were on β-blockers. Twelve patients had 3-vessel disease, and 1 had 2-vessel disease. No ischemic symptoms or DE evidence of ischemia were noted on follow-up.
Myocardial Function of Biopsied Segments
A total of 26 segments were biopsied: 5 had normal resting function (thickening fraction >30%) and served as control, 18 were hypokinetic, and 3 were akinetic. Of the 21 dysfunctional segments, 12 had a biphasic response, of which 9 (75%) recovered rest function. Of the 9 segments that had a worsening response to DE, only 3 (33%) showed recovery of rest function (P<0.05). Mean EF improved from 31±3% to 38±4% late after surgery (P=0.007), and NYHA class improved from 2.7±0.2 to 1.9±0.2 (P=0.01).
Myocardial Cytokines Versus Myocardial Function and Viability
As shown in Figure 1A, myocardial TNF-α mRNA was barely detectable in normal segments (1.2±0.2 copies of mRNA per million copies of β-actin, n=5). The number of copies of myocardial TNF-α was higher in segments with a biphasic response on DE (16.5±3.6, n=12) and was highest in segments with worsening response (ischemia without contractile reserve; 54.6±8.8, n=9; ANOVA P<0.001). Similarly, myocardial NOS2 mRNA levels were lowest in normal segments, intermediate in segments with contractile reserve, and highest in ischemic myocardium without contractile reserve (244.1±52.6 versus 1053.8±166.5 versus 3384.3±247.3, respectively; P<0.001). Results were similar if the analysis used recovery of function at 3 months as the criterion for viability (Figure 1B; P<0.001).
Relation of Serum TNF-α and Nitrite to Extent of Viability and Changes in Ventricular Function
Preoperative serum TNF-α ranged from 2.6 to 10.3 pg/mL and nitrite from 113.3 to 685.2 μmol/L. After revascularization, a significant decrease in serum TNF-α and nitrite levels was observed (TNF-α, 7.1±0.7 versus 5.5±1 pg/mL, P=0.02; nitrite, 449.3±56.5 versus 378.7±69.3 μmol/L, P=0.015). The change in EF from baseline to after revascularization related significantly to the changes in serum TNF-α and nitrite (Figure 2A). Although baseline serum TNF-α or nitrite levels bore no relationship to the likelihood of overall recovery of global left ventricular function, the extent of ischemic viable myocardium (number of biphasic segments) on the preoperative DE predicted the changes in serum TNF-α and nitrite (r=−0.92, P<0.001, Figure 2B) and the change in EF (r=0.9, P<0.001, data not shown).
We demonstrate that cardioinhibitory cytokines are regionally increased in HM. The levels of myocardial cytokines in HM are intermediate between those of normal myocardium, where they are barely detectable, and those seen in myocardium with less viability, where they are markedly upregulated. Serum cytokines decline after revascularization of viable myocardium. The magnitude of this decline correlates well with both the improvement in EF and the extent of viability.
Pathophysiology of Hibernating Myocardium
Although the precise mechanisms of HM are not fully understood, few broad pathophysiological categories are likely to be operational. The severity and duration of chronic hypoperfusion have been shown to determine the degree of structural alterations (myocyte degeneration, fibrosis) and the likelihood and speed of functional recovery after revascularization.1,2⇓ We have shown that there is a regional upregulation of α-adrenoceptor density and a downregulation of β-adrenoceptor density in ischemic dysfunctional myocardium.3 The present study shows, for the first time, an increase in regional cardioinhibitory cytokines in HM. The pattern of elevation in the gene expression for TNF-α and NOS2 was similar to that of the adrenergic receptors, namely, that the levels in HM are intermediate between those of normal myocardium and dysfunctional myocardium with minimal residual viability. Although it is not possible from our study to directly ascribe the resting dysfunction and presence or absence of viability to these changes in cytokine gene expression, several lines of evidence support this possibility. First, TNF-α and NOS2 are known to be upregulated in ischemia and in heart failure and to have negative inotropic effects.4 Second, TNF-α can induce the expression of NOS2 in cardiac myocytes, which in turn is partly responsible for its negative inotropic effects.5 Third, we have previously observed that overexpression of TNF-α in animal models leads to a phenotype that recapitulates that of ischemic cardiomyopathy with ventricular dilatation, depressed contractility, and an increase in collagen content that is related to a TNF-α–mediated increase in transforming growth factor-β.10 Along the same lines, Elsasser et al showed that the increased collagen content of HM is also related to the increased expression of transforming growth factor-β.11 Taken together, this suggests that upregulation of TNF-α in HM may play a role in mediating the increased fibrosis and depressed contractility. Fourth, NO is important in HM because it decreases oxygen consumption and preserves calcium sensitivity without an energy cost during ischemia.12 Finally, although it was not possible to obtain a follow-up biopsy to study the effects of revascularization on myocardial cytokine gene expression, we observed that serum levels of TNF-α and nitrite declined after revascularization and that the magnitude of this change correlated inversely with the change in EF and the number of viable segments determined before revascularization. Thus, it is likely that the chronic hypoperfusion seen in these dysfunctional myocardial regions drives the expression of these genes. The fact that differences in TNF-α and NOS2 expression are seen in the same heart indicates that factors in the local milieu, such as regional hypoperfusion, rather than global factors, such as ventricular filling pressure, may be more important in regulating regional cytokine gene expression.
Myocardial Cytokines and the Adrenergic System
Recent studies in animal models and in patients have established a link between sympathetic stimulation and cytokine expression.13 Release of norepinephrine is increased in dog hearts with HM.14 Ischemic cardiomyopathy is accompanied by sympathetic overdrive and a compensatory decrease in myocardial β-receptor density.3 Norepinephrine has also been shown to induce NOS2 gene expression in animal hearts. These effects are blocked by β-blockers.15 Given that sympathetic stimulation can upregulate cardioinhibitory cytokines in the setting of heart failure, this cross-talk between the adrenergic system and the cytokine system may play a role in the depressed contractility seen in HM. Along the same lines, TNF-α impairs β-adrenergic signal transduction and attenuates the increase in contractility in response to catecholamines.13 These findings are consistent with our observations that TNF-α levels were modestly increased in regions of viable myocardium with contractile reserve after dobutamine stimulation and markedly elevated in regions without contractile reserve. Previous work from our laboratory and others has suggested that mild elevations in these cytokines may protect against ischemia.16 In addition, although high doses of NO are negatively inotropic through the cGMP pathway, lower doses may actually preserve the contractile response through a cAMP mechanism.17 Thus, it is conceivable that whereas a modest elevation in cytokines may serve as a compensatory response to preserve viability and protect from cell death in the face of ischemia, a more pronounced elevation may be detrimental by promoting fibrosis, metabolic derangements, and loss of contractile reserve.
This study was supported by an investigator-initiated grant from the John S. Dunn Sr Trust Fund. Dr Dinesh Kalra is the recipient of an American College of Cardiology/Merck Fellowship Award.
Guest editor for this article was Shahbudin H. Rahimtoola, MD, University of Southern California, Los Angeles, Calif.
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