Tricyclic antidepressants directly depress human myocardial mechanical function independent of effects on the conduction system.

OBJECTIVES: To measure the effect of tricyclic antidepressant drugs (TCAs) on human myocardial contractility. METHODS: Human atrial tissue was obtained during cardiac bypass surgery. The tissue was harvested, suspended in a Tyrode buffer at 37 degrees C, and perfused with a 95%/5% oxygen-carbon dioxide mixture. Developed force was continuously measured using a force transducer and recorded by computer. After an equilibration period, escalating doses of amitriptyline or desipramine were added to the bath. All strips were exposed to the following five concentrations of each drug: 0 (control) 0.4, 4, 40, and 400 microM. The results for each experiment were expressed as the difference between the developed force measured prior to the addition of each concentration of drug and the developed force measured after a 30-minute exposure to the drug. RESULTS: Desipramine decreased the developed force by 27%, 49%, and 74% at concentrations of 0.4, 40, and 400 microM, respectively. Amitriptyline decreased the developed force by 38% at the 40-microM concentration and by 89% at the 400-microM concentration. Untreated strips retained 94% of baseline developed force at 150 minutes. CONCLUSIONS: Tricyclic antidepressants depress human myocardial function in a dose-dependent fashion independent of the effects on the cardiac conduction system. While previous work has demonstrated the effect of therapies for the reversal of impaired cardiac conduction following TCA poisoning, to the best of the authors' knowledge, no reports have documented the effects of therapy on direct myocardial depression. Additional therapies targeted at reversing the direct cardiodepressive effects of TCA may improve outcome following TCA poisoning.

Exogenous calcium preconditions myocardium from patients taking oral sulfonylurea agents.

We have previously reported that atrial trabeculae from patients taking oral sulfonylurea hypoglycemic agents cannot be preconditioned by transient ischemia, which may, in part, explain the increased cardiovascular mortality historically associated with the use of these agents (J. C. Cleveland et al., 1997, Circulation 96, 29-32). Recently, we reported that clinically accessible and acceptable exogenous Ca(2+) pretreatment protects human atrial trabeculae from subsequent ischemia (B. S. Cain et al., 1998, Ann. Thoracic Surg. 65, 1065-1070). It remains unknown whether this preconditioning strategy could confer protection to trabeculae from patients taking oral sulfonylurea drugs. We therefore hypothesized that exogenous Ca(2+) confers ischemic protection to trabeculae from patients taking oral sulfonylureas. Human atrial trabeculae were suspended in organ baths and field stimulated at 1 Hz, and force development was recorded. Following 90 min equilibration, trabeculae from patients taking oral sulfonylurea agents (n = 6 patients) were subjected to ischemia/reperfusion (I/R; 45/120 min) with or without Ca(2+) (1 mM increase x 5 min) 10 min prior to I/R. I/R decreased postischemic human myocardial contractility in trabeculae from patients on oral hypoglycemics to 15.3 +/- 2.0% baseline developed force (%BDF). Ca(2+) pretreatment increased postischemic human myocardial developed force to 35.3 +/- 2.9 %BDF in these patients (P < 0.05 vs I/R, ANOVA and Bonferroni/Dunn). We conclude that atrial muscle from patients taking oral hypoglycemic agents can be preconditioned with exogenous Ca(2+). This therapy may offer a clinically relevant means to precondition the myocardium of diabetics taking oral hypoglycemic agents prior to clinical interventions such as coronary angioplasty or cardiac bypass.

Tumor necrosis factor-alpha and interleukin-1beta synergistically depress human myocardial function.

OBJECTIVE: Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta have been implicated in the pathogenesis of myocardial dysfunction in ischemia-reperfusion injury, sepsis, chronic heart failure, viral myocarditis, and cardiac allograft rejection. Although circulating TNF-alpha and IL-1beta are both often elevated in septic shock, it remains unknown whether TNF-alpha or IL-1beta are the factors induced during sepsis that directly depress human myocardial function, and if so, whether the combination synergistically depresses myocardial function. Furthermore, the mechanism(s) by which these cytokines induce human myocardial depression remain unknown. We hypothesized the following: a) TNF-alpha and IL-1beta directly depress human myocardial function; b) together, TNF-alpha and IL-1beta act synergistically to depress human myocardial function; and c) inhibition of ceramidase or nitric oxide synthase attenuates myocardial depression induced by TNF-alpha or IL-1beta by limiting proximal cytokine signaling or production of myocardial nitric oxide (NO). DESIGN: Prospective, randomized, controlled study. SETTING: Experimental laboratory in a university hospital. SUBJECTS: Freshly obtained human myocardial trabeculae. INTERVENTIONS: Human atrial trabeculae were obtained at the time of cardiac surgery, suspended in organ baths, and field simulated at 1 Hz, and the developed force was recorded. After a 90-min equilibration, TNF-alpha (1.25, 12.5, 125, or 250 pg/mL for 20 mins), IL-1beta (6.25, 12.5, 50, or 200 pg/mL for 20 mins), or TNF-alpha (1.25 pg/mL) plus IL-1beta (6.25 pg/mL) were added to the bath, and function was measured for the subsequent 100 mins after the 20-min exposure. To assess the roles of the sphingomyelin and NO pathways in TNF-alpha and IL-1beta cross-signaling, the ceramidase inhibitor N-oleoyl ethanolamine (1 microM) or the NO synthase inhibitor N(G)-monomethyl-L-arginine (10 microM) was added before TNF-alpha (125 pg/mL) or IL-1beta (50 pg/mL). MEASUREMENTS AND MAIN RESULTS: TNF-alpha and IL-1beta each depressed human myocardial function in a dose-dependent fashion (maximally depressing to 16.2 + 1.9% baseline developed force for TNF-alpha and 25.7 + 6.3% baseline developed force for IL-1beta), affecting systolic relatively more than diastolic performance (each p < .05). However, when combined, TNF-alpha and IL-1beta at concentrations that did not individually result in depression (p > .05 vs. control) resulted in contractile depression (p < .05 vs. control). Inhibition of myocardial sphingosine or NO release abolished the myocardial depressive effects of either TNF-alpha or IL-1beta. CONCLUSIONS: TNF-alpha and IL-1beta separately and synergistically depress human myocardial function. Sphingosine likely participates in the TNF-alpha and IL-1beta signal leading to human myocardial functional depression. Therapeutic strategies to reduce production or signaling of either TNF-alpha or IL-1beta may limit myocardial dysfunction in sepsis.

Inhibition of myocardial TNF-alpha production by heat shock. A potential mechanism of stress-induced cardioprotection against postischemic dysfunction.

Overproduction of tumor necrosis factor-alpha (TNF-alpha) contributes to cardiac dysfunction associated with systemic or myocardial stress, such as endotoxemia and myocardial ischemia/reperfusion (I/R). Heat shock has been demonstrated to enhance cardiac functional resistance to I/R. However, the protective mechanisms remain unclear. The purpose of this study was to determine: (1) whether cardiac macrophages express heat shock protein 72 (HSP72) after heat shock, (2) whether induced cardiac HSP72 suppresses myocardial TNF-alpha production during I/R, and (3) whether preservation of postischemic myocardial function by heat shock is correlated with attenuated TNF-alpha production during I/R. Rats were subjected to heat shock (42 degrees C for 15 min) and 24 h recovery. Immunoblotting confirmed the expression of cardiac HSP72. Immunofluorescent staining detected HSP72 in cardiac interstitial cells including resident macrophages rather than myocytes. Global I/R caused a significant increase in myocardial TNF-alpha. The increase in myocardial TNF-alpha was blunted by prior heat shock and the reduced myocardial TNF-alpha level was correlated with improved cardiac functional recovery. This study demonstrates for the first time that heat shock induces HSP72 in cardiac resident macrophages and inhibits myocardial TNF-alpha production during I/R. These observations suggest that inhibition of myocardial TNF-alpha production may be a mechanism by which HSP72 protects the heart against postischemic dysfunction.

Cardiotrophin-1 attenuates endotoxin-induced acute lung injury.

Cardiotrophin-1 (CT-1) is a recently discovered member of the gp130 cytokine family, which includes IL-6, IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Recent evidence suggests that, like other members of this family, CT-1 may possess anti-inflammatory properties. We hypothesized that in vivo CT-1 administration would attenuate endotoxin (ETX)-induced acute lung injury. We studied the effects of CT-1 (100 microgram/kg ip, 10 min prior to ETX) in a rat model of ETX-induced acute lung injury (Salmonella typhimurium lipopolysaccharide, 20 mg/kg ip). Six hours after ETX, lungs were harvested for determination of neutrophil accumulation (myeloperoxidase, MPO, assay) and lung edema (wet-to-dry weight ratio). Mechanisms of pulmonary vasorelaxation were examined in isolated pulmonary artery rings at 6 h by interrogating endothelium-dependent (response to acetylcholine) and endothelium-independent (response to sodium nitroprusside) relaxation following alpha-adrenergic (phenylephrine)-stimulated preconstriction. CT-1 abrogated the endotoxin-induced lung neutrophil accumulation: 2.3 +/- 0.2 units MPO/g wet lung (gwl) vs 6. 3 +/- 0.3 units MPO/gwl in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Similarly, CT-1 prevented ETX-induced lung edema: wet-to-dry-weight ratio, 4.473 +/- 0.039 vs 4.747 +/- 0.039 in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Endotoxin caused significant impairment of both endothelium-dependent and -independent pulmonary vasorelaxation, and CT-1 attenuated this injury. Thus, cardiotrophin-1 possesses significant anti-inflammatory properties in a model of endotoxin-induced acute lung injury.

Calcium preconditioning, but not ischemic preconditioning, bypasses the adenosine triphosphate-dependent potassium (KATP) channel.

BACKGROUND: Recent evidence has implicated the KATP channel as an important mediator of ischemic preconditioning (IPC). Indeed, patients taking oral sulfonylurea hypoglycemic agents (i.e., KATP channel inhibitors) for treatment of diabetes mellitus are resistant to the otherwise profoundly protective effects of IPC. Unfortunately, many cardiopulmonary bypass patients, who may benefit from IPC, are chronically exposed to these agents. Calcium preconditioning (CPC) is a potent form of similar myocardial protection which may or may not utilize the KATP channel in its mechanism of protection. The purpose of this study was to determine whether CPC may bypass the KATP channel in its mechanism of action. If so, CPC may offer an alternative to IPC in patients chronically exposed to these agents. METHODS: Isolated rat hearts (n = 6-8/group) were perfused (Langendorff) and received KATP channel inhibition (glibenclamide) or saline vehicle 10 min prior to either a CPC or IPC preconditioning stimulus or neither (ischemia and reperfusion, I/R). Hearts were subjected to global warm I/R (20 min/40 min). Postischemic myocardial functional recovery was determined by measuring developed pressure (DP), coronary flow (CF), and compliance (end diastolic pressure, EDP) with a MacLab pressure digitizer. RESULTS: Both CPC and IPC stimuli protected myocardium against postischemic dysfunction (P < 0.05 vs I/R; ANOVA with Bonferroni/Dunn): DP increased from 52 +/- 4 (I/R) to 79 +/- 2 and 83 +/- 4 mmHg; CF increased from 11 +/- 0.7 to 17 +/- 2 and 16 +/- 1 ml/min; and EDP decreased (compliance improved) from 50 +/- 7 to 27 +/- 5 and 31 +/- 7 mmHg. However, KATP channel inhibition abolished protection in hearts preconditioned with IPC (P < 0.05 vs IPC alone), but not in those preconditioned with CPC (P > 0.05 vs CPC alone). CONCLUSIONS: (1) Both IPC and CPC provide similar myocardial protection; (2) IPC and CPC operate via different mechanisms; i.e., IPC utilizes the KATP channel whereas CPC does not; and (3) CPC may offer a means of bypassing the deleterious effects of KATP channel inhibition in diabetic patients chronically exposed to oral sulfonylurea hypoglycemic agents.

Therapeutic strategies to reduce TNF-alpha mediated cardiac contractile depression following ischemia and reperfusion.

Recent evidence has implicated proinflammatory mediators such as TNF- alpha in the pathophysiology of ischemia-reperfusion (I/R) injury. Clinically, serum levels of TNF-alpha are increased after myocardial infarction and after cardiopulmonary bypass. Each of these represent clinically relevant instances of cardiac I/R injury. We and others have recently reported that TNF-alpha is produced by the heart following experimental I/R in animals and that TNF-alpha directly decreases animal and human myocardial contractility in a dose dependent fashion. Thus, strategies to reduce or neutralize myocardial TNF- alpha production should conceptually decrease myocardial contractile dysfunction following I/R. The purposes of this manuscript are: 1) to explore the clinical and experimental instances of I/R injury in which TNF-alpha is elevated, 2) to review the molecular mechanisms of TNF- alpha induced contractile dysfunction, 3) to examine both experimental and clinical strategies of reducing myocardial TNF-alpha production, and 4) to determine the influence of reducing post-I/R TNF-alpha on cardiac contractile function in both animals and man.

p38 MAPK inhibition decreases TNF-alpha production and enhances postischemic human myocardial function.

INTRODUCTION: TNF-alpha is a proinflammatory cytokine implicated in myocardial dysfunction following ischemia/reperfusion (I/R). I/R results in myocardial production of TNF-alpha and TNF-alpha suppresses myocardial contractility. p38 mitogen-activated protein kinase (MAPK) is a redox-sensitive protein kinase involved in intracellular signaling leading to TNF-alpha production. It remains unknown if the human heart produces TNF-alpha after I/R and, if so, whether p38 MAPK is involved. HYPOTHESIS: p38 MAPK inhibition enhances human myocardial post-I/R contractile function by inhibition of myocardial TNF-alpha production. METHODS: Human atrial trabeculae were suspended in organ baths, field simulated at 1 Hz, and force development was recorded. Following a 90-min equilibration, trabeculae were exposed to a p38 MAPK inhibitor (SB 203580, 1 microM) or vehicle (each n = 6) prior to simulated ischemia (45 min hypoxia, substrate-free, rapid pacing at 3 Hz) followed by 120 min reoxygenation. Myocardial TNF-alpha levels were measured by ELISA at end reoxygenation. RESULTS: I/R increased human myocardial TNF-alpha levels from 26.9 +/- 9.3 to 83.9 +/- 19.2 pg/g wet tissue (P < 0.05 perfusion vs I/R; ANOVA Bonferroni/Dunn), while p38 MAPK inhibition decreased post-I/R myocardial TNF-alpha levels to 32.3 +/- 8.0 pg/g wet tissue (P > 0.05 p38 MAPK inhibition vs I/R). p38 MAPK inhibition improved postischemic force development from 18.5 +/- 2.1 to 37.0 +/- 2.0% baseline developed force (%BDF; P < 0.05 I/R vs p38 MAPK inhibition). CONCLUSIONS: (1) The human heart produces TNF-alpha after I/R, (2) p38 MAPK mediates myocardial I/R-induced TNF-alpha production, (3) p38 MAPK inhibition limits functional impairment after I/R, and (4) inhibition of ischemia-induced TNF-alpha production may represent a potent therapeutic strategy for improving myocardial function after angioplasty, coronary bypass, or heart transplantation.

Protein kinase C in normal and pathologic myocardial states.

Recent evidence has implicated protein kinase C (PKC) as an important mediator underlying multiple aspects of myocardial function. We and others have recently reported that PKC is involved in Ca2+-induced inotropy, in mediating myocardial preconditioning by diverse stimuli both in animals and humans, and in the signaling processes which lead to the production of proinflammatory mediators (cytokines). The purposes of this manuscript are to explore the role of PKC in normal myocardial contraction, the role of PKC in mediating protection, the role of PKC in inflammation, and the importance of inflammation regarding myocardial injury.

Clinical L-type Ca(2+) channel blockade prevents ischemic preconditioning of human myocardium.

Although Ca(2+) channel blockers are commonly used to control both blood pressure and angina in patients with coronary artery disease, clinical trials have associated the use of L-type Ca(2+) channel blockers with increased cardiovascular mortality. Recent evidence has implicated Ca(2+) entry through the L-type Ca(2+) channel during transient ischemia as a proximal stimulus for ischemic preconditioning (IPC) in experimental animals. We therefore hypothesized that clinical L-type Ca(2+) channel blockade prevents IPC in human myocardium. Human atrial trabeculae were suspended in organ baths, field simulated at 1 Hz, and force development was recorded. Following 90 min equilibration, trabeculae from control patients and patients taking L-type Ca(2+) channel blockers were subjected to simulated ischemia/reperfusion (I/R: 45/120 min) with or without 5 min of simulated ischemia (IPC stimulus) prior to I/R. IPC increased post-ischemic developed force in control patients from 14.6+/-2.6 to 43.1+/-3.5% baseline developed force (%BDF P<0.05 I/R vs IPC). Whereas IPC failed to increase post-ischemic developed force in myocardium from patients taking L-type Ca(2+) channel blockers (15. 1+/-1.9 vs 16.6+/-1.7 %BDF, P>0.05 L-type I/R v L-type IPC). We conclude that: (1) atrial muscle can be preconditioned by transient ischemia; (2) atrial muscle from patients taking L-type Ca(2+) channel blockers cannot be preconditioned by transient ischemia; and (3) the increased cardiovascular mortality historically associated with the use of Ca(2) channel blockers in patients with coronary artery disease may be, in part, due to the pharmacological inhibition of ischemic preconditioning.

Ischemic preconditioning decreases postischemic myocardial tumor necrosis factor-alpha production. Potential ultimate effector mechanism of preconditioning.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion (I/R) injury, sepsis, chronic heart failure, and cardiac allograft rejection. Cardiac resident macrophages, infiltrating leukocytes, and cardiomyocytes themselves produce TNF-alpha. Although adenosine reduces macrophage TNF-alpha production and protects myocardium against I/R, it remains unknown whether ischemic preconditioning, which is mediated by adenosine, decreases postischemic myocardial TNF-alpha production. METHODS AND RESULTS: Isolated rat hearts were crystalloid perfused with the Langendorff method and subjected to global, normothermic I/R (20/40 minutes), with or without prior transient ischemic preconditioning (5 minutes) or adenosine pretreatment. Postischemic cardiac TNF-alpha (ELISA) and function were determined (Langendorff). I/R increased cardiac TNF-alpha and impaired myocardial function. Ischemic preconditioning or adenosine decreased myocardial TNF-alpha and improved postischemic functional recovery. Sequestration of myocardial TNF-alpha (TNF binding protein) during the I/R experiments similarly improved postischemic myocardial function. CONCLUSIONS: This study constitutes the initial demonstration that in addition to its other beneficial effects, preconditioning decreases postischemic myocardial TNF-alpha, an autocrine contributor to postischemic myocardial dysfunction. Reduced myocardial TNF-alpha production may represent the distal effector mechanism of preconditioning.

Human myocardial tissue TNFalpha expression following acute global ischemia in vivo.

Although human myocardial TNFalpha levels are increased during the ischemia associated with chonic heart failure, it remains unknown whether an acute global ischemic insult further increases TNFalpha expression in human cardiac myocytes. To study this, biopsies of human myocardium were obtained before and after cardiopulmonary bypass (in vivo acute global ischemia), and myocardial TNFalpha levels were determined by ELISA and cytotoxicity assay (WEHI-164 clone 13 cell line). TNFalpha was immunolocalized by immunohistochemistry. Results indicate that cardiopulmonary bypass induces an increase in human myocardial TNFalpha by both ELISA and cytotoxicity assays. Immunolocalization revealed that prior to cardiopulmonary bypass TNFalpha was located predominantly in the myocardial interstitial cells; however, following bypass, increased TNFalpha was observed in the cardiocytes themselves. Locally-produced myocardial TNFalpha may be an important contributor to myocardial functional depression and injury following acute ischemia. Targeted anti-TNFalpha therapy in the treatment of cardiac ischemic injury may further elucidate its clinical relevance.

Mechanisms of pH preservation during global ischemia in preconditioned rat heart: roles for PKC and NHE.

Ischemic preconditioning (PC) attenuates cardiac acidosis during global ischemia. This adaptation to ischemia is detectable before other better known indexes of PC are manifested. Clarification of the endogenous mechanisms may provide insights into how protein kinase C (PKC) signaling might be linked to altered intracellular biochemistry. 31P NMR studies of isolated, buffer-perfused rat heart were performed to determine whether functionally cardioprotective PC by cyclic ischemia (CI) and alpha1-adrenergic stimuli [phenylephrine (PE)] attenuated acidosis during ischemia and, if so, whether this 1) involves a PKC-dependent pathway and is due to 2) decreased glycolytic proton production, 3) an increase in proton buffering, or 4) proton extrusion. At the end of 20 min of global ischemia, both CI-PC (pH = 6.86 +/- 0.14) and PE-PC (pH = 6.90 +/- 0.13) attenuated end-ischemic acidosis (control pH = 6.54 +/- 0.1). PKC blockade with chelerythrine (Chel) prevented the attenuation of ischemic acidosis by PC stimuli (end-ischemic pH: CI + Chel, 6.43 +/- 0.06; PE + Chel, 6.17 +/- 0.17). End-ischemic lactate accumulation was decreased in CI-PC hearts (7.54 +/- 0.5 vs. control, 14.61 +/- 2.1 micromol/g wet wt) but not in those preconditioned through the alpha1-adrenergic receptor (12.25 +/- 0.9 micromol/g wet wt). Physiologically relevant buffers were not increased in the preconditioned groups. Blockade of the Na+/H+ exchanger [NHE; with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or HOE-694] eliminated the attenuation of ischemic acidosis seen with PC stimuli (pH: CI + EIPA, 6.5 +/- 0.1; PE + EIPA, 6.46 +/- 0.2; PE + HOE-694, 6.26 +/- 0.15; not significantly different from control). We conclude that CI and alpha1-adrenergic PC stimuli attenuate ischemic acidosis, and this may involve the cardiac amiloride-sensitive NHE. The signaling pathways of both these two stimuli appear to involve PKC.

Increased levels of myocardial IkappaB-alpha protein promote tolerance to endotoxin.

Endotoxin [lipopolysaccharide (LPS)] causes tumor necrosis factor-alpha (TNF-alpha)-mediated myocardial contractile depression. Tolerance to the cardiac toxicity of LPS can be induced by a prior exposure to LPS or by pretreatment with glucocorticoids. The mechanisms by which the myocardium acquires tolerance to LPS remain unknown. LPS causes phosphorylation and degradation of inhibitory kappaB-alpha (IkappaB-alpha), releasing nuclear factor-kappaB (NF-kappaB) to activate TNF-alpha gene transcription. We hypothesized that LPS induces supranormal synthesis of myocardial IkappaB-alpha protein and thus renders the myocardium tolerant to subsequent LPS. Rats were challenged with LPS after pretreatment with LPS, dexamethasone, or saline. In saline-pretreated rats, LPS caused a rapid decrease in myocardial IkappaB-alpha protein levels, activation of NF-kappaB, and increased TNF-alpha production. These events were followed by myocardial contractile depression. After the initial decrease in myocardial IkappaB-alpha, IkappaB-alpha protein levels rebounded to a level greater than control levels by 24 h. Dexamethasone pretreatment similarly increased myocardial IkappaB-alpha protein levels. In rats pretreated with either LPS or dexamethasone, myocardial IkappaB-alpha protein levels remained similar to control levels after LPS challenge. The preserved level of myocardial IkappaB-alpha protein was associated with diminished NF-kappaB activation, attenuated myocardial TNF-alpha production, and improved cardiac contractility. We conclude that LPS and dexamethasone upregulate myocardial IkappaB-alpha protein expression and that an increased level of myocardial IkappaB-alpha protein may promote cardiac tolerance to LPS by inhibition of NF-kappaB intranuclear translocation and myocardial TNF-alpha production.

Hydrogen peroxide induces tumor necrosis factor alpha-mediated cardiac injury by a P38 mitogen-activated protein kinase-dependent mechanism.

BACKGROUND: Oxidant stress caused by ischemia or endotoxemia induces myocardial dysfunction and cardiomyocyte death; however, mechanisms responsible remain unknown. We hypothesized that hydrogen peroxide (H2O2) induces myocardial dysfunction and cardiomyocyte death via P38 mitogen-activated protein kinase (MAPK)-mediated myocardial tumor necrosis factor (TNF) production. METHODS: Langendorff perfused rat hearts (6/group) were subjected to oxidant stress (H2O2 infusion; 300 mmol/L x 80 minutes), with and without prior infusion of a specific P38 kinase MAPK inhibitor (P38i = 1 mmol/L/min x 5 minutes) or TNF neutralization (20 mg TNF binding protein (BP)/min x 80 minutes). Developed pressure (DP), coronary flow, and end-diastolic pressure were continuously recorded. Myocardial creatine kinase (CK) loss was measured in the coronary effluent, and tissue TNF was measured in myocardial homogenates. RESULTS: Eighty minutes of H2O2 infusion induced a 6.5-fold increase in myocardial TNF production, which was associated with a 70% decrease in DP and increase in CK loss. P38 MAPK inhibition or TNF-BP decreased myocardial TNF production, cardiomyocyte death, and myocardial dysfunction. CONCLUSIONS: These results demonstrate that H2O2 alone induces myocardial TNF production. P38 MPAK is an oxidant-sensitive enzyme that mediates oxidant-induced myocardial TNF production, cardiac dysfunction, and cardiomyocyte death.

Human SERCA2a levels correlate inversely with age in senescent human myocardium.

OBJECTIVES: This study sought to characterize functional impairment after simulated ischemia-reperfusion (I/R) or Ca2+ bolus in senescent human myocardium and to determine if age-related alterations in myocardial concentrations of SERCA2a, phospholamban, or calsequestrin participate in senescent myocardial dysfunction. BACKGROUND: Candidates for elective cardiac interventions are aging, and an association between age and impairment of relaxation has been reported in experimental animals. Function of the sarcoplasmic reticulum resulting in diastolic dysfunction could be dysregulated at the level of cytosolic Ca2+ uptake by SERCA2a, its inhibitory subunit (phospholamban), or at the level of Ca2+ binding by calsequestrin. METHODS: Human atrial trabeculae from 17 patients (45-75 years old) were suspended in organ baths, field simulated at 1 Hz, and force development was recorded during I/R (45/120 min). Trabeculae from an additional 12 patients (53-73 years old) were exposed to Ca2+ bolus (2-3 mmol/L bath concentration). Maximum +/- dF/dt and the time constant of force decay (tau) were measured before and after I/R or Ca2+ bolus and related to age. SERCA2a, phospholamban, and calsequestrin from 12 patients (39-77 years old) were assessed by immunoblot. RESULTS: Functional results indicated that maximum +/-dF/dt and tau were prolonged in senescent (>60 years) human myocardium after I/R (p < 0.05). Calcium bolus increased the maximum +/-dF/dt and decreased tau in younger, but not older patients (p < 0.05). SERCA2a and the ratio of SERCA2a to either phospholamban or calsequestrin were decreased in senescent human myocardium (p < 0.05). CONCLUSIONS: Senescent human myocardium exhibits decreased myocardial SERCA2a content with age, which may, in part, explain impaired myocardial function after either I/R or Ca2+ exposure.

TNF-alpha and myocardial depression in endotoxemic rats: temporal discordance of an obligatory relationship.

Exogenous tumor necrosis factor-alpha (TNF-alpha) induces delayed myocardial depression in vivo but promotes rapid myocardial depression in vitro. The temporal relationship between endogenous TNF-alpha and endotoxemic myocardial depression is unclear, and the role of TNF-alpha in this myocardial disorder remains controversial. Using a rat model of endotoxemia not complicated by shock, we sought to determine 1) the temporal relationship of changes in circulating and myocardial TNF-alpha with myocardial depression, 2) the influences of protein synthesis inhibition or immunosuppression on TNF-alpha production and myocardial depression, and 3) the influence of neutralization of TNF-alpha on myocardial depression. Rats were treated with lipopolysaccharide (LPS, 0.5 mg/kg ip). Circulating and myocardial TNF-alpha increased at 1 and 2 h, whereas myocardial contractility was depressed at 4 and 6 h. Pretreatment with cycloheximide or dexamethasone abolished the increase in circulating and myocardial TNF-alpha and preserved myocardial contractile function. Similarly, treatment with TNF binding protein immediately after LPS prevented myocardial depression. We conclude that endogenous TNF-alpha mediates delayed myocardial depression in endotoxemic rats and that inhibition of TNF-alpha production or neutralization of TNF-alpha preserves myocardial contractile function in endotoxemia.