Carmine V. Oddis

Interleukin-6 (IL-6) as a mediator of stunned myocardium

Abstract Proinflammatory cytokines are a class of secretory polypeptides that are synthesized and released locally by macrophages, leukocytes and endothelial cells in response to injury. 1–4 Reperfusion of ischemic myocardium is associated with infiltration by leukocytes and macrophages that may be responsible for transient depression in myocardial contractility (“stunned myocardium”). 1,2 We have recently demonstrated that certain cytokines (IL-2, IL-6, and TNFα) have direct, reversible negative inotropic effects in isolated hamster papillary muscle preparations. 5 The observed inotropic effects of these cytokines raised the possibility that they contribute to myocardial stunning. A direct effect of cytokines on human myocardium in concentrations achievable clinically would be necessary to further support this hypothesis. The frequency of myocardial stunning after cardiopulmonary bypass provided an ideal opportunity to assay the levels as well as the inotropic effects of these cytokines in human hearts. Written, informed consent was obtained from patients undergoing elective aortocoronary bypass grafting in accordance with the guidelines of the University of Pittsburgh Institutional Review Board for Biomedical Research. The elaboration of cytokines by pulmonary macrophages in response to injury suggested that pulmonary venous blood might be a rich source of inflammatory cytokines after cardiopulmonary bypass. 6 Plasma levels of TNFα and IL-6 in pulmonary venous effluents were measured from 7 patients immediately before and after uncomplicated coronary artery bypass grafting.

Cytokine induction during cardiac surgery: analysis of TNF-alpha expression pre- and postcardiopulmonary bypass.

Cytokines are soluble mediators that possess both intra‐and intercellular signaling properties. Their function has been investigated most thoroughly in the context of immune reactions. Cytokines, however, form an important component of the inflammatory response to trauma and are found in elevated concentrations following cardiopulmonary bypass (CPB). We were, therefore, interested in studying mRNA expression for the cytokine tumor necrosis factor alpha (TNF‐α) in patients undergoing cardiac surgery. White cells isolated from the circulation of seven patients before and after CPB were assayed for the expression of mRNA with TNF‐α specific primers using the polymerase chain reaction. Message for TNF‐α was found in all patients, with the highest values demonstrated in patients whose CPB times exceeded 1 1/2 hours. Enhanced message expression was seen as early as 1 hour after the start of CPB. Genes responsible for cytokine (TNF‐α) expression and production are activated during cardiac surgery.

Regulation off constitutive nitric oxide synthase activity by the human heart

We and others have provided indirect evidence for the presence of a constitutive nitric oxide synthase (cNOS) in the mammalian heart. We now provide more direct evidence for the regulation of a myocardial cNOS in the hearts of patients undergoing elective cardiopulmonary bypass (CPB). cNOS enzyme activity was demonstrable in both cytosolic (8.3 +/- 0.02 pmol/min/mg) and membrane (11.1 +/- 0.4 pmol/min/mg) preparations derived from human atrial pectinate muscles obtained at the time of CPB (n = 6). Plasma nitrite (NO2-) + plasma nitrate (NO3-) levels from the beating hearts of patients before bypass were reduced from 146 +/- 33 to 5.1 +/- 50 pmol/min/g after cardiac arrest during CPB (n = 23; p < 0.002 by Students t test). Thus, the human myocardium constitutively produces nitric oxide that is regulated by the contractile state of the heart.

Cytokines and nitric oxide synthase inhibitor as mediators of adrenergic refractoriness in cardiac myocytes

We have previously proposed that pro-inflammatory cytokines and nitric oxide (NO) contributed to reversible myocardial depression in patients with sepsis and congestive heart failure. Sepsis and heart failure are also associated with refractoriness to beta-adrenoceptor agonists. Therefore, the chronotropic effects of cytokines and the NO synthase inhibitor, NG-methyl-L-arginine (NMA), on beta-adrenoceptor stimulation of neonatal cardiac myocytes were studied. Tumor necrosis factor alpha, interleukin-1 beta and interleukin-6 but not interleukin-4 or interleukin-5 significantly enhanced spontaneous beating rates compared to untreated myocytes in serum-free media for 48 h (P < 0.01; n = 12 for each). NMA also significantly enhanced spontaneous beating rates (P < 0.01; n = 12 for each). Only interleukin-1 beta treatment resulted in significant nitrite production, immunohistochemical staining for inducible nitric oxide synthase and detection of inducible NO synthase messenger RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). However, tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, and NMA each completely blocked the positive chronotropic effects of the beta-adrenoceptor agonist, isoproterenol (P < 0.01; n = 12 for each). These findings are most consistent with an inducible NO synthase-independent effect of cytokines and NMA on the chronotropic responses of neonatal cardiac myocytes to beta-adrenoceptor stimulation. This effect of cytokines and NMA on adrenergic signaling may involve a myocardial constitutive NO synthase or an NO-independent mechanism.

Positive inotropic effect of acetylcysteine in cardiomyopathic Syrian hamsters

Several laboratories have provided indirect evidence that the myocardium of the cardiomyopathic Syrian hamster (CMH) is chronically ischemic on the basis of microvascular spasm. We previously reported evidence supporting a defect in the ryanodine-sensitive sarcoplasmic reticulum calcium release channel (SRCRC) in CMH. A relation between alterations in SRCRC and chronic ischemia has not yet been explored. A potential mechanism could be the effects of changes in redox state on thiol groups. Thiol reagents have previously been shown to regulate calcium release from SRCRC. Accordingly, we studied the inotropic effects of the sulfhydryl donors, acetylcysteine (AC), cysteine, and cystine in CMH. AC was a positive inotrope in isolated papillary muscles prepared from CMH, but not F1B controls (F1B) (p<0.01). No significant differences were noted in inotropic responses to cysteine or cystine. AC blunted the response of CMH>F1B control papillary muscle preparations to stimulation frequency (p<0.01). The actual tension generated (in mg/mm2) by CMH was no longer different than F1B with addition of AC (10-3 M), ryanodine (10-8 M), or verapamil (5 × 10-7 M). These findings are consistent with a defect in SRCRC in CMH. This defect may be primary or may provide a novel mechanism for hibernating myocardium owing to chronic ischemia.

Verapamil regulation of a defective SR release channel in the cardiomyopathic Syrian hamster.

The Bio 14.6 Cardiomyopathic Syrian Hamster (CMH) has an autosomal recessive disease characterized by intracellular calcium overload, cardiac and skeletal myopathies and premature death from congestive heart failure. Early treatment of these animals with the calcium antagonist, verapamil (V), prevents the development of the disease. We have previously provided evidence supporting a specific defect in the ryanodine-sensitive SR calcium release channel (SRCRC) in CMH. We now provide physiologic and biochemical evidence that V modulates SRCRC. Papillary muscles prepared from F1B control hamsters (F1B) revealed an enhanced inotropic responsiveness to V and ryanodine (R) with age, not seen with CMH. CMH papillary muscles demonstrated paradoxical positive inotropic effects of V and R not shared with F1B. The positive inotropic effects of V and R were not additive. V enhanced the affinity (decreased KD) of [3H]ryanodine binding to cardiac membranes. Thus, V may prevent the overt manifestations of genetic disease in CMH by modulating a defective ryanodine-sensitive SR release channel.

Inotropic effects of calcium antagonists in the cardiomyopathic Syrian hamster

The inotropic responses of papillary muscles isolated from the BIO 14.6 cardiomyopathic Syrian hamster were compared with those of age-matched F, B controls. Length-tension curves revealed the preservation of contractile function at 1–2 months of age and significant loss of function at 4–6 months of age. Papillary muscles prepared from 4–6-month-old myopathic hamsters were significantly less sensitive to increasing frequency of stimulation than were controls (p < 0.05). There were no differences in the responses to nifedipine, Bay K 8644, diltiazem, or gallopamil. Only verapamil demonstrated a biphasic inotropic response in the cardiomyopathic hamster with a low-dose positive inotropic effect (131 ± 4% at 4 ± 2 × 10 -7 M) and a 50-fold higher IC50 for negative inotropy compared with F1B controls (200 ± 30 vs. 4.0 ± 1.0 μM). Verapamil is also a less potent negative inotrope in 1–2-month-old myopathic papillary muscles compared with controls (IC50, 280 ± 70 vs. 32 ± 10 μM; p < 0.05). These inotropic effects are not shared by the other calcium channel modulators studied (i.e., nifedipine, Bay K 8644, diltiazem, gallopamil). These findings do not support the presence of a functional defect in the sarcolem-mal L-type calcium channel in the cardiomyopathic Syrian hamster. The mechanism of action of verapamil in the cardiomyopathic Syrian hamster remains to be elucidated.

Bay K 8644 is a negative inotrope in the presence of arachidonic acid but not eicosapentaenoic acid

Summary: The inotropic and radioligand binding characteristics of arachidonic acid (AA) and the fish oil derivative, eicosapentaenoic acid (EPA), were studied. A A alone was a concentration-dependent negative inotrope in isolated hamster papillary muscles. The negative inotropic effect of AA was enhanced 500-fold by the coadministration of indomethacin (cyclo-oxygenase inhibitor) and caffeic acid (lipoxygenase inhibitor). The dihydropyridine calcium channel agonist Bay K 8644 (BK) was a concentration-dependent positive inotrope. BK in the presence of AA was a concentration-dependent negative inotrope. The negative inotropic effect of BK in the presence of AA was blocked by caffeic acid but not indomethacin. EPA alone was a concentration-dependent negative inotrope. EPA had no effect on the positive inotropic effect of BK. Scatchard analyses of [3H]nitrendipine (dihydropyridine) binding to cardiac membranes in the presence of AA or EPA resulted in a decrease in the KD (-1/slope) with no effect on Bmax (x intercept). Kinetic studies further revealed that both AA and EPA enhanced the off rate of [3H]nitrendipine binding. These data suggest that both AA and EPA mediate similar direct negative inotropic effects through cardiac sarcolemmal calcium channels. A unique effect on E-C coupling by AA, not shared by EPA, is mediated through the lipoxygenase pathway (leukotrienes). AA and its eicosanoid products may play critically important roles in regulating myocardial contractility during acute and chronic myocardial ischemia. Differences between AA and EPA may contribute to the beneficial effects of dietary fish oils enriched in EPA.

Myocardial ischemia, stunning and hibernation: blood blow, metabolism and pathophysiology mechanism

It was first shown in the opened chest dog model that ischemia followed by reperfusion was associated with a period of depressed myocardial contractility [1–4]. The clinical implications of these experimental observations were not fully appreciated until sophisticated imaging techniques enabled clinicians to non-invasively assess myocardial contractility and viability in patients [5, 6]. Quite surprisingly, it became increasingly apparent that a number of clinical conditions resulted in reversible depression of myocardial contractility. Reversible myocardial depression following reperfusion of ischemic myocardium was documented in patients following myocardial infarction, cardiopulmonary bypass, thrombolytic therapy and coronary angioplasty [5, 6]. This condition has been referred to as’ stunned’ myocardium. Coronary artery bypass grafting (CABG) of chronically ischemic myocardium was also shown to result in improvement in the left ventricular ejection fraction in some patients [7, 8]. The term ‘hibernating myocardium’ has been adopted to refer to reversibly depressed chronically ischemic myocardium that remains viable but does not contract [9].

Regulation of [3H]nitrendipine binding by phospholipases A2 and C through direct and GTP-sensitive mechanisms

We compared the effects of Phospholipases A2, C, B and D on [3H]nitrendipine binding to hamster cardiac membranes, in the absence and presence of ATP or GTP. Phospholipase A2, competitively inhibited [3H]nitrendipine binding to hamster cardiac membranes unchanged by ATP or GTP (Ki = 5 ng/ml); as evidenced by complete and reversible displacement of [3H]nitrendipine binding and increase in KD on Scatchard analyses. Phospholipase C also completely inhibited [3H]nitrendipine binding to hamster cardiac membranes (Ki = 5 micrograms/ml) with a decrease in Bmax and no change in KD on Scatchard analyses. The addition of GTP alone inhibited the PLC effect in EGTA-treated membranes. The addition of GTP with either CaCl2 or ATP or both resulted in an equal and opposite enhancement of the PLC effect. Phospholipases B and D had no effect on [3H]nitrendipine binding. These data support: (1) Direct effect of PLA2 on dihydropyridine binding. (2) Indirect regulation of dihydropyridine binding by Phospholipase C through a GTP and ATP-sensitive mechanism.