1 Myocardial dysfunction during septic shock is associated with enhanced production of cytokines such as interleukin-1p and tumour necrosis factor-x (TNF-x). These cytokines depress cardiac mechanical function by a mechanism which is not well defined.2 Bacterial endotoxin or cytokines cause the expression of Ca2"-independent nitric oxide (NO) synthase in cardiac myocytes, vascular endothelial cells and endocardial endothelial cells, causing enhanced production of NO. As NO has negative inotropic actions on cardiac muscle, we tested the sum effects of IL-lp plus TNF-a in the intact heart to determine whether enhanced expression of NO synthase activity in the cells that comprise the heart is involved in cardiac depression associated with cytokine stimulation.3 Rat isolated working hearts perfused with IL-1p plus TNF-a showed a markedly greater depression in contractile function, measured as cardiac work, after 2 h of perfusion compared with time-matched control hearts. The depressant action of IL-ip plus TNF-a was first apparent after 1 h of perfusion; no early (15min) cardiac depressant actions were seen. 4 The competitive inhibitor of Ca2"-dependent and Ca2"-independent NO synthases, N0-nitro-Larginine methyl ester (L-NAME, 3#M) when given concurrently with IL-lp plus TNF-x prevented the loss in contractile function such that these hearts after 2h of perfusion had similar function to time-matched controls. L-NAME did not acutely reverse the loss of contractile function in hearts exposed for 2 h to IL-1 plus TNF-x. The protective action of L-NAME in the presence of cytokines was concentration-dependent and was not seen at a higher concentration (10 pM) due to the significant reduction in coronary flow observed at this concentration.5 In contrast, when L-NAME (3 1M) was given in the absence of IL-lP plus TNF-x it depressed contractile function over the 2 h perfusion period by significantly reducing coronary flow. 6 Inhibition of protein synthesis with cycloheximide (Cx) abolished the loss in function that occurred over 2h in both control and IL-il plus TNF-a-treated hearts.7 Inducible, Ca2"-independent NO synthase activity was not observed in freshly isolated hearts but was observed in control hearts perfused for 2 h in vitro and was doubled in hearts perfused with IL-IP plus TNF-a. Cx prevented the expression of Ca2"-independent NO synthase in both control and cytokine-treated hearts.8 In summary, these results suggest that the depression of myocardial function by IL-lP plus TNF-a is mediated, at least in part, by induction of Ca2"-independent NO synthase activity in the heart.

. Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats. Am J Physiol Heart Circ Physiol 283: H1108-H1115, 2002. First published May 16, 2002 10.1152/ajpheart.00549. 2001.-Free radicals have been implicated in the etiology of cardiac dysfunction during sepsis, but the actual species responsible remains unclear. We studied the alterations in myocardial nitric oxide (NO), superoxide, and peroxynitrite generation along with cardiac mechanical function and efficiency in hearts from lipopolysaccharide (LPS)-treated rats. Six hours after LPS (4 mg/kg ip) or saline (control) treatment, hearts were isolated and perfused for 1 h with recirculating Krebs-Henseleit buffer and paced at 300 beats/min. Cardiac work, O2 consumption, and cardiac efficiency were markedly depressed in LPS hearts compared with controls. Plasma nitrate/nitrite level was elevated in LPS rats, and ventricular NO production was enhanced as measured by electron spin resonance spectroscopy, Ca 2ϩ -independent NO synthase (NOS) activity, and inducible NOS immunohistochemistry. Ventricular superoxide production was also enhanced in LPS-treated hearts as seen by lucigenin chemiluminescence and xanthine oxidase activity. Increased nitrotyrosine staining (immunohistochemistry) and higher lipid hydroperoxides levels were also detected in LPS-treated hearts, indicating oxygen radical-induced stress. Enhanced generation of both NO and superoxide, and thus peroxynitrite, occur in dysfunctional hearts from endotoxemic rats. sepsis; cardiac dysfunction; nitric oxide; superoxide and peroxynitrite SEPTIC SHOCK is characterized by severe hypotension with profound vasodilatation and multiple organ failure resulting from systemic release of inflammatory cytokines in response to an infective organism (35). Depression of myocardial contractility is a well-documented feature of septic shock (15, 34) despite the fact that assessment of intrinsic cardiac function is complicated by a marked increase in heart rate and decreased preload and afterload. Data from both clinical (32) and experimental (29) studies indicate the presence of genuine myocardial dysfunction when assessed independently of changes in hemodynamics. However, the etiological mechanism(s) of cardiac dysfunction in sepsis is not well understood, but various circulating and/or locally produced mediators have been implicated (for review see Ref. 19).Evidence from our laboratory (39, 40) and from others (2, 3) suggests that exposure of animal hearts or isolated cardiac myocytes to bacterial endotoxin (lipopolysaccharides, LPS) or proinflammatory cytokines enhanced nitric oxide (NO) generation via induction of NO synthase (iNOS). The production of large amounts of NO by this enzyme may have detrimental effects on the myocardium (2, 9, 39). On the other hand, NO may be cardioprotective (37) and may also act as an antioxidant molecule (45).Recent studies have indicated that many of the deleterious effects of NO are mediated by peroxynitrite; this powerful oxidant is generated from a f...

Proinflammatory cytokines (interleukin-1β, tumor necrosis factor-α, and interferon-γ; Cytomix) depress myocardial contractile work partially by stimulating expression of inducible nitric oxide (NO) synthase (iNOS). Because NO and peroxynitrite inhibit myocardial O2 consumption (MV˙o 2), we examined whether this mechanism contributes to reduced cardiac work. In control isolated working rat hearts, cardiac work was stable for 60 min, followed by a decline from 60 to 120 min, without change in MV˙o 2. Cardiac efficiency (work/MV˙o 2) was therefore reduced from 60 to 120 min. Cytomix shortened the onset (within 20–40 min) and enhanced the depression in cardiac work and efficiency and inhibited MV˙o 2 after 80 min. Mercaptoethylguanidine (MEG), an iNOS inhibitor and peroxynitrite scavenger, or the glucocorticoid dexamethasone (Dex) abolished the effects of Cytomix. iNOS expression was increased 10-fold by Cytomix and abolished by Dex but not MEG. That cytokine-induced depression in cardiac work precedes the reduction in MV˙o 2 suggests, at least in the early response, that NO and/or peroxynitrite may not impair heart function by inhibiting mitochondrial respiration but reduce the heart’s ability to utilize ATP for contractile work.

We examined the mechanism of the time- and protein synthesis-dependent decline in cardiac mechanical function in isolated working rat hearts. Hearts were perfused with Krebs-Henseleit buffer for 120 min in the presence or absence of the protein synthesis inhibitor cycloheximide (CX; 10 μM). Cardiac work remained stable for 60 min and then spontaneously decreased during 60–120 min of perfusion. This was accompanied by an increase in myocardial inducible nitric oxide synthase (iNOS) and xanthine oxidase (XO) activities and enhanced dityrosine formation in the perfusate, an indicator of peroxynitrite generation. CX markedly attenuated the loss in contractile function and prevented the increase in iNOS and XO activities and dityrosine level. Despite the decline in cardiac work in control hearts, the coupling between tricarboxylic acid (TCA) cycle activity and oxygen consumption remained constant in both groups. ATP, creatine phosphate, and glycogen levels were not different between control and CX groups and did not differ over 120 min of perfusion. We concluded that the delayed and spontaneous loss in myocardial mechanical function in isolated working rat hearts is 1) attenuated by CX treatment, 2) accompanied by a concomitant increase in both iNOS and XO activities and peroxynitrite generation in the heart, and 3) not dependent on a direct impairment in myocardial ATP production, myocardial oxygen consumption, or TCA cycle acetyl-CoA production but may be due to an inefficiency of the heart to utilize ATP for contractile work.

Lysophospholipid accumulation has been implicated in the pathogenesis of irreversible injury during myocardial ischemia and reperfusion. Plasmalogens (phospholipids with a vinyl-ether bond in the sn-1 position) account for more than 50%1 of total myocardial sarcolemmal and sarcoplasmic reticulum phospholipids. Accumulation of plasmalogen choline and ethanolamine lysophospholipids (lysoplasmenylcholine and lysoplasmenylethanolamine) or the effects of exogenous fatty acids on lysoplasmalogen accumulation during ischemia and reperfusion have not been examined. Isolated working rat hearts perfused with buffer containing either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate were subjected to aerobic, ischemic, or ischemia/reperfusion protocols. Levels of lysoplasmenylcholine and lysoplasmenylethanolamine were quantified using a two-stage high-performance liquid chromatographic technique. In hearts perfused with glucose alone, no significant differences in levels of lysoplasmenylcholine or lysoplasmenylethanolamine were seen during ischemia or reperfusion. In fatty acid-perfused hearts, however, significant accumulation of lysoplasmenylethanolamine occurred during reperfusion but not during ischemia (723+ 112, 734+83, and 1,394± 193 nmol/g dry wt for aerobic, ischemic, and ischemic/reperfused hearts, respectively; p<0.05 for ischemic/reperfused hearts versus aerobic or ischemic hearts). Lysoplasmenylcholine levels after ischemia and reperfusion did not differ significantly from aerobic values, regardless of whether fatty acids were present or absent from the perfusate. Aerobic and ischemic/reperfused rabbit hearts, in the presence of fatty acid, showed a similar profile in their lysoplasmalogen content. We conclude that differential lysoplasmenylethanolamine accumulation occurs during myocardial reperfusion when exogenous fatty acid concentrations are high. This may reflect the selective action of fatty acid intermediates on the metabolism of lysoplasmenylethanolamines. The role of lysoplasmalogens in producing the depressed mechanical recovery of fatty acid-perfused hearts after ischemia/reperfusion remains to be