Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.

Dilated cardiomyopathy is a severe pathology of the heart with poorly understood etiology. Disruption of the gene encoding the negative immunoregulatory receptor PD-1 in BALB/c mice, but not in BALB/c RAG-2-/- mice, caused dilated cardiomyopathy with severely impaired contraction and sudden death by congestive heart failure. Affected hearts showed diffuse deposition of immunoglobulin G (IgG) on the surface of cardiomyocytes. All of the affected PD-1-/- mice exhibited high-titer circulating IgG autoantibodies reactive to a 33-kilodalton protein expressed specifically on the surface of cardiomyocytes. These results indicate that PD-1 may be an important factor contributing to the prevention of autoimmune diseases.

Sodium/calcium exchange modulates intracellular calcium overload during posthypoxic reoxygenation in mammalian working myocardium. Evidence from aequorin-loaded ferret ventricular muscles.

We tested the hypothesis that the intracellular Ca2+ overload of ventricular myocardium during the period of posthypoxic reoxygenation is mediated by transsarcolemmal Ca2+ influx via Na+/Ca2+ exchange. In aequorin-loaded, ferret right ventricular papillary muscles, blockers of the sarcolemmal and the sarcoplasmic reticulum Ca2+ channels, slowed the Cai2+ transient, producing a convex ascent during membrane depolarization, followed by a concave descent during repolarization. The magnitude of the Cai2+ transient was affected by changes in the membrane potential, Nai+, Nao+, and Cao2+, and was blocked by Ni2+, or dichlorbenzamil. The calculated Na+/Ca2+ exchange current was in the reverse mode (Ca2+ influx) during the ascending phase of the Cai2+ transient, and was abruptly switched to the forward mode (Ca2+ efflux) at repolarization, matching the time course of the Cai2+ transient. During hypoxic superfusion, the Cai2+ transient was abbreviated, which was associated with a shorter action potential duration. In contrast, immediately after reoxygenation, the Cai2+ transient increased to a level greater than that of the control, even though the action potential remained abbreviated. This is the first demonstration on a beat-to-beat basis that, during reoxygenation, Ca2+ influx via Na+/Ca2+ exchange is augmented and transports a significant amount of Ca2+ into the ventricular myocardial cell. The activation of the exchanger at the time of reoxygenation appears to be mediated by Nai+ accumulation, which occurs during hypoxia.

Digoxin reduces beta-adrenergic contractile response in rabbit hearts. Ca(2+)-dependent inhibition of adenylyl cyclase activity via Na+/Ca2+ exchange.

Whereas mobilization of intracellular Ca2+ stimulates neuronal adenylyl cyclase via Ca2+/calmodulin, mobilized Ca2+ directly inhibits adenylyl cyclase in other tissues. To determine the physiologic role of the Ca(2+)-dependent interaction between Na+/Ca2+ exchange and beta-adrenergic signal transduction in the intact heart, digoxin (0.3 mg/kg) was administered intravenously in rabbits. 30 min after the administration, digoxin impaired the peak left ventricular dP/dt response to dobutamine infusions by up to 38% as compared with control rabbits. This impairment was not caused by changes in either beta-adrenergic receptor number or in the functional activity of stimulatory guanine nucleotide-binding protein. It was associated with 33-36% reductions in basal and stimulated adenylyl cyclase activities. Animals treated with calcium gluconate (20 mg/kg/min for 30 min) also demonstrated similar reductions in adenylyl cyclase activities. In addition, increasing the free Ca2+ concentration progressively inhibited adenylyl cyclase activity in the control, digoxin-treated, and calcium gluconate-treated sarcolemma preparations in vitro. Moreover, digoxin and calcium gluconate pretreatment blunted the increase in cAMP in myocardial tissue after dobutamine infusion in vivo. Thus, digoxin rapidly reduces beta-adrenergic contractile response in rabbit hearts. This reduction may reflect an inhibition of adenylyl cyclase by Ca2+ mobilized via Na+/Ca2+ exchange.

Treatment of virus-induced myocardial injury with a novel immunomodulating agent, vesnarinone. Suppression of natural killer cell activity and tumor necrosis factor-alpha production.

Controversy still exists concerning the therapy for viral myocarditis which manifests a wide variety of clinical symptoms. Vesnarinone, a quinolinone derivative that was developed as a positive inotropic agent with complex actions, including phosphodiesterase inhibition and cation channel modification, has recently been confirmed to improve the prognosis of patients with chronic heart failure. However, the precise mechanism of this beneficial effect is not yet clearly understood. In this study, using a murine model of acute viral myocarditis resulting from encephalomyocarditis virus infection, survival and myocardial damage were markedly improved by treatment with vesnarinone. In contrast, survival was not improved by treatment with amrinone, a phosphodiesterase inhibitor. Although vesnarinone did not inhibit viral replication or protect myocytes from viral direct cell injury, it did inhibit the increase in natural killer cell activity after viral infection. On the other hand, amrinone failed to inhibit natural killer cell activity. Both vesnarinone and amrinone suppressed the production of tumor necrosis factor-alpha. Therefore, we postulate that vesnarinone exerted its beneficial effects through an inhibition of natural killer cell activity, and that it serves as an immunomodulator providing new therapeutic possibilities for the treatment of viral myocarditis and/or immunological disorders.

Enhanced expression of superoxide dismutase messenger RNA in viral myocarditis. An SH-dependent reduction of its expression and myocardial injury.

The oxygen free radical system has been reported to be activated by influenza virus infection in the lungs. However, the involvement of oxygen radicals in viral myocarditis is still unknown. Captopril, an angiotensin-converting enzyme (ACE) inhibitor and potent free radical scavenger with a sulfhydryl group, was effective for the treatment of viral myocarditis, while enalapril, an ACE inhibitor without a sulfhydryl group, was not effective against acute myocarditis. In this study, we investigated the role of oxygen radicals in the pathogenesis of viral myocarditis and the therapeutic effects of agents with a sulfhydryl group. 4-wk-old BALB/c mice were inoculated with the encephalomyocarditis virus, and treated with captopril or N,2-mercapto-propionyl glycine (MPG), a sulfhydryl-containing amino acid derivative without ACE inhibiting property, from days 4 to 14. On day 14, captopril and MPG significantly improved survival of mice and myocardial injury (necrosis, cellular infiltration, and calcification) in a dose-dependent manner compared with the infected control group. Thus, captopril and MPG were effective for the treatment of virus-induced myocarditis. Furthermore, a striking induction of manganese superoxide dismutase (Mn-SOD) and copper/zinc SOD (Cu/Zn-SOD) mRNAs in infected hearts was found (8-13-fold for Mn-SOD and 4-11-fold for Cu/Zn-SOD) when compared with age-matched uninfected mice hearts. MPG completely inhibited the increase of both mRNAs, even when treatment was started on day 4. Thus, oxygen radicals may play an important role in the pathogenesis of viral myocarditis, and a therapeutic approach by eliminating oxygen radicals seems possible.

Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts.

The cardiac ATP-sensitive potassium (KATP) channel is thought to be a complex composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the sulfonylurea receptor (SUR2). This channel is activated during myocardial ischemia and protects the heart from ischemic injury. We examined the transcriptional expression of these genes in rats with myocardial ischemia. 60 min of myocardial regional ischemia followed by 24-72 h, but not 3-6 h, of reperfusion specifically upregulated Kir6.1 mRNA not only in the ischemic (approximately 2.7-3.1-fold) but also in the nonischemic (approximately 2.0-2.6-fold) region of the left ventricle. 24 h of continuous ischemia without reperfusion also induced an increase in Kir6.1 mRNA in both regions, whereas 15-30 min of ischemia followed by 24 h of reperfusion did not induce such expression. In contrast, mRNAs for Kir6.2 and SUR2 remained unchanged under these ischemic procedures. Western blotting demonstrated similar increases in the Kir6.1 protein level both in the ischemic (2.4-fold) and the nonischemic (2.2-fold) region of rat hearts subjected to 60 min of ischemia followed by 24 h of reperfusion. Thus, prolonged myocardial ischemia rather than reperfusion induces delayed and differential regulation of cardiac KATP channel gene expression.

Fc receptor-mediated inhibitory effect of immunoglobulin therapy on autoimmune giant cell myocarditis: concomitant suppression of the expression of dendritic cells.

In the present study, the mechanisms and importance of the Fc portion of immunoglobulin in experimental giant cell myocarditis were examined. Giant cell myocarditis was induced in rats by immunization of porcine cardiac myosin. Human intact immunoglobulin (1 g. kg(-1). d(-1)) or F(ab')(2) fragments of human immunoglobulin (1 g. kg(-1). d(-1)) were administered intraperitoneally daily on days 1 to 21. Intact immunoglobulin administration significantly ameliorated myocarditis, but F(ab')(2) fragments did not. The ribonuclease protection assay revealed that therapy with intact immunoglobulin, but not F(ab')(2) fragments, suppressed the mRNA expressions of inflammatory and proinflammatory cytokines. Immunohistochemical analysis showed that therapy with intact immunoglobulin, but not F(ab')(2) fragments, suppressed dendritic cell (DC) expression during both the early and the subsequent fulminant phases. Moreover, the early treatment of intact immunoglobulin until the 11th day or 14th day, when the expression of DCs was completely suppressed, ameliorated myocarditis. However, the late treatment of intact immunoglobulin beginning on day 15, when the expression of DCs had already been completed, failed to ameliorate the condition. An in vitro study showed that intact immunoglobulin, but not F(ab')(2) fragments, suppressed the lipopolysaccharide-induced interleukin-1beta production associated with the downregulation of CD32 antigen (Fcgamma receptor II) expression. Thus, intact immunoglobulin therapy markedly suppressed myocarditis as a result of Fc receptor-mediated anti-inflammatory action, and the suppression of the disease was associated with the suppression of DCs, ie, the suppression of the initial antigen-priming process in experimental giant cell myocarditis.

Calcineurin pathway is required for endothelin-1-mediated protection against oxidant stress-induced apoptosis in cardiac myocytes.

Endothelin-1 (ET-1) acts not only as a growth-promoting peptide but also as a potent survival factor against myocardial cell apoptosis. However, the signaling pathways leading to myocardial cell protection by ET-1 are poorly understood. Using a culture system of primary cardiac myocytes derived from neonatal rats, we show in the present study that ET-1 almost completely blocked the hydrogen peroxide-induced increase in the percentage of TdT-mediated dUTP-biotin nick-end labeling-positive myocytes. Apoptosis inhibition by ET-1 was confirmed by cytofluorometric analysis as well as by examination of the ladder formation, morphological features, and caspase-3 cleavage. We have found that ET-1 converts the nuclear factor of activated T lymphocytes (NFATc) in cardiac myocytes into high-mobility forms and translocates cytoplasmic NFATc to the nuclei. In addition, ET-1 stimulates the interaction between NFATc and the cardiac-restricted zinc-finger protein GATA4 in these cells. The immunosuppressants cyclosporin A and FK506, which antagonize calcineurin, negated the inhibitory effect of ET-1 on apoptosis. Calcineurin activation de novo was sufficient to inhibit hydrogen peroxide-induced apoptosis. ET-1 induced the expression of an antiapoptotic protein bcl-2 in cardiac myocytes in a cyclosporin A-dependent manner, but it did not alter the expression of bax. Cyclosporin A also attenuated the ET-1-stimulated transcription of the bcl-2 gene in these cells. These findings demonstrate that the calcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress-induced apoptosis in cardiac myocytes.

Treatment of experimental viral myocarditis with interleukin-10.

BACKGROUND: The T helper cell type 2-associated cytokine interleukin (IL)-10 has a variety of immunomodulatory properties. However, the effects of the cytokine on viral myocarditis remain unclear. METHODS AND RESULTS: We studied the effects of recombinant human IL-10 (rhIL-10) fully active on mouse cells in a murine experimental model of acute viral myocarditis caused by the encephalomyocarditis virus (EMCV). Four-week-old DBA/2 mice were inoculated with EMCV (day 0). rhIL-10 (10 microg/mouse) was administered once daily, starting on day 0, and control mice received vehicle only. Survival rates were determined on day 14. Myocardial histopathology, cytokine levels in the heart by ELISA assay, and myocardial virus concentration were examined on day 6, and the expression levels of myocardial inducible nitric oxide synthase (iNOS) mRNA were measured by competitive polymerase chain reaction. The 14-day survival in mice treated with rhIL-10 was significantly higher (80%) than in the control group (30%, n=10 in each, P<0.05). rhIL-10 treatment significantly attenuated myocardial lesions and suppressed tumor necrosis factor-alpha and IL-2 in the heart. rhIL-10 treatment had little effect on myocardial virus concentration. The expression levels of myocardial iNOS mRNA were significantly decreased in the group treated with rhIL-10 (8.6+/-4.7 amol/mg total RNA in treated versus 26.5+/-7.1 amol/mg total RNA in control mice, P<0.05). CONCLUSIONS: These findings provide new insights into the in vivo effects of IL-10 on viral infection and suggest a therapeutic effect of IL-10 on viral myocarditis.

Contribution of endothelin-1 to myocardial injury in a murine model of myocarditis: acute effects of bosentan, an endothelin receptor antagonist.

BACKGROUND: Endothelin (ET) is one of the most important contributing factors in the pathophysiology of cardiovascular diseases. However, little is known about its role in myocarditis. METHODS AND RESULTS: Four-week-old DBA/2 mice were inoculated with the encephalomyocarditis virus. Expression levels of ET-converting enzyme-1 (ECE-1) and prepro-ET-1 mRNA were significantly increased at 7 and 14 days after virus inoculation. Plasma and myocardial ET-1 levels were significantly higher in infected than noninfected mice between 5 and 14 days after virus inoculation. Immunohistochemical analyses revealed that not only endothelial cells and myocytes but also infiltrating mononuclear cells produced ET-1 protein at 7 days. Oral bosentan, a mixed ET-1 receptor antagonist, was administered after virus inoculation in doses of 0 (control group), 10, or 100 mg. kg(-1). d(-1), and the animals were killed on day 14. Mean heart weight/body weight ratios were 8.3+/-1.8 versus 11.2+/-2.4 versus 10. 8+/-2.4 in the bosentan 100 mg. kg(-1). d(-1) versus 10 mg. kg(-1). d(-1) versus control groups, respectively (P<0.05). Corresponding histological scores for myocardial necrosis were 2.0+/-0.2 versus 2. 9+/-0.3 versus 3.0+/-0.4 (P<0.05), and cellular infiltration scores were 2.3+/-0.3 versus 2.9+/-0.4 versus 3.3+/-0.4 (P<0.05). Animals killed on day 5 had significantly smaller necrotic areas after treatment with bosentan 100 mg. kg(-1). d(-1) than the group treated with a lower dose or the control group, despite the absence of differences in virus titers. CONCLUSIONS: This study suggests that ET-1 plays an important pathophysiological role in viral myocarditis. Treatment with bosentan had a cardioprotective effect without modifying viral replication.

Endothelin-1 has a unique oxygen-saving effect by increasing contractile efficiency in the isolated rat heart.

BACKGROUND: The effect of endothelin (ET)-1 on cardiac energetics is not fully understood. METHODS AND RESULTS: In isolated, coronary-perfused rat hearts, we measured left ventricular contractility index (E(max)), pressure-volume area (PVA), and myocardial oxygen consumption (MVO(2)) before and after administration of ET-1 (1x10(-)(9) mol/L). ET-1 increased E(max) by 48+/-16% (P<0.01) and the total MVO(2) by 24+/-11% (P<0.01). The MVO(2)-PVA relations were linear both before and after ET-1 (r>0.99). ET-1 shifted MVO(2)-PVA upward, increasing the MVO(2) intercept by 24+/-13%. At the same time, ET-1 decreased the slope (S), with 1/S (contractile efficiency) being 46+/-5% before and 56+/-5% after ET-1 (P<0.01). ET-1-induced increases in E(max) and in contractile efficiency were abolished by an ET(A) receptor blocker (S-0139) but not by an ET(B) blocker (BQ-788). Although high [Ca(2+)] perfusion increased E(max) and the intercept to the same extent as ET-1, it did not change S. N(G)-Nitro-L-arginine (an inhibitor of nitric oxide synthase) increased the coronary perfusion pressure as much as ET-1, but S again remained unchanged. Dimethylamyloride (Na(+)/H(+) exchanger inhibitor) partially blocked the positive inotropic effect of ET-1 but not the ET-1-induced increase in the contractile efficiency. CONCLUSIONS: Agonistic effects of ET-1 on the ET(A) receptor economized the chemomechanical conversion efficiency of the left ventricular unit myocardium by a mechanism independent of the Na(+)/H(+) exchanger. This unique oxygen-saving effect of ET-1 may play an adaptive role in the failing myocardium, in which local accumulation of ET-1 is present.

FTY720, a new immunosuppressant, promotes long-term graft survival and inhibits the progression of graft coronary artery disease in a murine model of cardiac transplantation.

Background-Effective immunosuppression is a critical determinant of organ and patient survival in cardiac transplantation. The present study was designed to determine the potency of FTY720, a new synthesized immunosuppressant, and examine its clinical potential as an immunosuppressant. Methods and Results-Hearts of DBA/2 mice were transplanted heterotopically in C57BL/6 mice. Recipients were treated with oral FTY720 in doses of 0.3, 1, 3, or 10 mg. kg(-1). d(-1) or with 40 mg. kg(-1). d(-1) of cyclosporin A (CsA) as a comparative treatment. The median graft survival time (MST) was significantly prolonged by treatment with FTY720 10 mg. kg(-1). d(-1). MST was not prolonged by FTY720 1 mg. kg(-1). d(-1) or CsA. However, FTY720 1 mg. kg(-1). d(-1) combined with CsA 40 mg. kg(-1). d(-1) resulted in a significant prolongation of MST. Histopathological studies performed 5 days after transplantation demonstrated remarkable suppression of inflammatory response by treatment with FTY720 10 mg. kg(-1). d(-1). Interleukin (IL)-2 and interferon (IFN)-gamma production was not suppressed; however, cytotoxic T lymphocyte activity was strongly suppressed in vitro. In addition, IL-2-stimulated T-cell proliferation and class I and class II MHC antigen expression on IFN-gamma-stimulated macrophages were strongly inhibited by FTY720. Histopathological studies 60 days after transplantation (DBA/2-B10.D2) demonstrated a beneficial effect on graft atherosclerosis. Conclusions-FTY720 promoted long-term cardiac graft survival and strongly inhibited the progression of graft atherosclerosis. These observations suggest that FTY720 has a promising clinical potential in cardiac transplantation.

alpha- and beta-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes.

BACKGROUND: The apoptosis of cardiac myocytes may play a role in the development of heart failure. Norepinephrine is one of the factors activated in heart failure and can induce myocardial cell apoptosis in culture. However, it is unknown if alpha- and beta-adrenergic pathways coordinately or differentially regulate apoptosis and if this apoptotic pathway uses common or cell type-specific apoptotic signals. METHODS AND RESULTS: We stimulated cultured neonatal rat cardiac myocytes with an alpha(1)-adrenergic agonist (PE, phenylephrine), a beta-adrenergic agonist (isoproterenol [Iso]) or a membrane-permeable cAMP analogue (8-Br-cAMP) in serum-free conditions for 48 hours. Iso and 8-Br-cAMP markedly increased the number of TUNEL-positive cells (%TUNEL-positive nuclei >40%) compared with saline stimulation (<10%). DNA fragmentation was also confirmed by ladder formation in agarose gels. Apoptotic myocytes were characterized by cell shrinkage and nuclear condensation, consistent with morphological features of apoptosis. The Iso-induced apoptosis was almost completely inhibited by the protein kinase A-specific inhibitor KT5720. In contrast, PE inhibited 8-Br-cAMP-induced myocardial cell apoptosis. The apoptosis-inhibitory effect by PE was negated by the alpha(1)-adrenergic receptor antagonist prazosin and the MEK-1-specific inhibitor PD098059. Interestingly, although 8-Br-cAMP markedly induced apoptosis in cardiac myocytes, it completely blocked serum depletion-induced apoptosis in PC12 cells, a rat pheochromocytoma cell line. CONCLUSIONS: These findings indicate that alpha- and beta-adrenergic pathways differentially regulate myocardial cell apoptosis. The results also suggest that a cAMP- protein kinase A pathway is necessary and sufficient for beta-adrenergic agonist-induced apoptosis and that this apoptotic pathway is not functional in other cell types, for example, PC12 cells.

The role of endothelin-converting enzyme-1 in the development of alpha1-adrenergic-stimulated hypertrophy in cultured neonatal rat cardiac myocytes.

BACKGROUND: Accumulating evidence suggests that the local synthesis of endothelin-1 (ET-1) plays a role in the development of heart failure in vivo. We investigated the role of endothelin-converting enzyme-1 (ECE-1), which mediates the conversion of big ET-1 to mature ET-1, in the development of alpha1-adrenergic-stimulated hypertrophy in cultured neonatal rat cardiac myocytes. METHODS AND RESULTS: Phenylephrine (PE) induced the expression of ET-1 in rat cardiac myocytes and accelerated the conversion of big ET-1 to ET-1. The ECE-1 mRNA levels were markedly increased 3 hours after PE stimulation (3.6-fold compared with saline stimulation, P<0.005). A specific ECE-1 antagonist, FR901533, inhibited the PE-stimulated increase in protein synthesis rate by 45% (P<0.05). As genetic markers for the hypertrophic response, FR901533 inhibited the PE-stimulated transcriptional activities of the 3.5-kb beta-myosin heavy chain promoter by 79% (P<0.01) but did not affect that of the 3.4-kb atrial natriuretic factor (ANF) promoter. In Bio14.6 Syrian cardiomyopathic hamsters, ventricular ET-1 and ANF mRNA levels did not correlate at 2 different stages. CONCLUSIONS: ET-1-independent pathways may mediate activation of the ANF gene program in ventricular myocytes both in vitro and in vivo. These results also indicate that the conversion of big ET-1 to ET-1 in rat cardiac myocytes is required for the development of alpha1-adrenergic-stimulated hypertrophy and beta-myosin heavy chain gene transcription.

Differential effects of angiotensin II versus endothelin-1 inhibitions in hypertrophic left ventricular myocardium during transition to heart failure.

BACKGROUND: In view of their mutual crosstalk, the roles of angiotensin II (Ang II) and endothelin-1 (ET-1) in the myocardium are assumed to be synergistic and supplemental. METHODS AND RESULTS: In the phase of compensated left ventricular (LV) hypertrophy of Dahl salt-sensitive rats, Ang II peptide and the ACE mRNA in the LV were increased by 1.6- and 3.8-fold, respectively. In contrast, ET-1 peptide and the preproET-1 mRNA remained unchanged. In subsequent congestive heart failure (CHF), Ang II and ACE mRNA did not show further increases. But ET-1 and the mRNA were increased de novo by 5.3- and 4.1-fold, respectively. In ascending aorta-banded rats, the local activations of Ang II and ET-1 also showed a differential time course between LV hypertrophy and CHF. Long-term treatments of Dahl salt-sensitive rats with temocapril (an ACE inhibitor) and with bosentan (a mixed ET receptor blocker) equally improved long-term survival. Temocapril reduced the LV/body weight ratio and ameliorated LV fractional shortening. Conversely, although bosentan equally improved fractional shortening, it did not reduce the increase in LV mass. Combined treatment with these 2 drugs further ameliorated the animal's survival without additional decreases in systolic pressure. CONCLUSIONS: The pathophysiological roles in the myocardium during the transition to CHF differ qualitatively between Ang II and ET-1. Thus, long-term therapy with a combination of ACE inhibition and ET antagonism may provide a new approach for heart failure in humans.

Persistently increased serum concentrations of cardiac troponin t in patients with idiopathic dilated cardiomyopathy are predictive of adverse outcomes.

BACKGROUND: The measurement of serum concentrations of cardiac troponin T (TnT) is a simple, useful method to detect myocyte injury that may be repeated multiple times to follow patients without interobserver variability. METHODS AND RESULTS: Multiple measurements of TnT with a second-generation assay were performed in 60 patients with dilated cardiomyopathy confirmed by coronary angiography and endomyocardial biopsy between April 1996 and December 1999. Three evolutionary patterns of TnT concentrations were identified. Thirty-three patients had concentrations of TnT <0.02 ng/mL throughout the follow-up period (group 1). The remaining 27 patients had high initial serum concentrations of TnT (>/=0.02 ng/mL). In 10 of these 27 patients, TnT decreased to <0.02 ng/mL during follow-up (group 2), whereas 17 had persistently high serum TnT concentrations despite being conventionally treated for chronic congestive heart failure (group 3). Although the initial echocardiographic left ventricular diastolic dimension (LVDd) and left ventricular ejection fraction (LVEF) were not significantly different among the 3 groups, follow-up echocardiography showed significantly decreased LVDd and increased LVEF in group 1 (each P:<0.01) and group 2 (each P:<0.05) compared with increased LVDd and decreased LVEF in group 3 (each P:<0.05). The cardiac event-free rate was significantly lower in group 3 than in groups 1 and 2 (each P:<0.001), and the survival rate was lower in group 3 than in group 1 (P:<0.05). CONCLUSIONS: Persistently increased TnT concentrations in dilated cardiomyopathy suggest ongoing subclinical myocyte degeneration associated with deterioration of the patients' clinical status.

Major racial differences in coronary constrictor response between japanese and caucasians with recent myocardial infarction.

BACKGROUND: Enhanced coronary vasomotion may contribute to acute coronary occlusion during the acute phase of myocardial infarction (AMI). Japanese have a higher incidence of variant angina than Caucasian patients, but racial differences in vasomotor reactivity early after AMI are controversial. METHODS AND RESULTS: The same team studied 15 Japanese and 19 Caucasian patients within 14 days of AMI by acetylcholine injection into non-infarct-related (NIRA) and infarct-related (IRA) coronary arteries followed by nitroglycerin. Incidence of vasodilation, vasoconstriction, spasm, and basal tone were assessed in proximal, middle, and distal segments after each drug bolus by quantitative angiography. Japanese patients had much lower cholesterol levels than Caucasians (183+/-59 versus 247+/-53 mg/dL, P<0.006) but showed a lower incidence of vasodilation (2% versus 9% of coronary segments) and a greater incidence of spasm after acetylcholine (47% versus 15% of arteries, P<0.00001). Incidence of spasm was higher in IRAs than in NIRAs in both populations (67% versus 39% and 23% versus 11%, respectively). Multivessel spasm was more common (64% versus 17%, P<0.02) and vasoconstriction of nonspastic segments was greater in Japanese patients (-23.4+/-14.9% versus -20.1+/-15.7%, P<0.02) in the presence of similar average basal coronary tone with respect to post-nitroglycerin dilation and of nonsignificant differences of coronary atherosclerotic score. CONCLUSIONS: Soon after AMI, Japanese patients exhibited a 3-fold-greater incidence of spasm and greater vasoconstriction of nonspastic segments after acetylcholine than Caucasians. The causes of such differences warrant further investigation because they may have relevant pathophysiological and therapeutic implications.

Anti-ischemic effect of a novel cardioprotective agent, JTV519, is mediated through specific activation of delta-isoform of protein kinase C in rat ventricular myocardium.

BACKGROUND: A new 1,4-benzothiazepine derivative, JTV519, has a strong protective effect against Ca(2+) overload-induced myocardial injury. We investigated the effect of JTV519 on ischemia/reperfusion injury in isolated rat hearts. METHODS AND RESULTS: At 30 minutes of reperfusion after 30-minute global ischemia, the percent recovery of left ventricular developed pressure was improved, and the creatine phosphokinase and lactate dehydrogenase leakage was reduced in a concentration-dependent manner when JTV519 was administered in the coronary perfusate both at 5 minutes before the induction of ischemia and at the time of reperfusion. The myocardial protective effect of JTV519 was completely blocked by pretreatment of the heart with GF109203X, a specific protein kinase C (PKC) inhibitor. In contrast, the effect of JTV519 was not affected by alpha(1)-, A(1)-, and B(2)-receptor blockers that couple with PKC in the cardiomyocyte. Both immunofluorescence images and immunoblots of JTV519-treated left ventricular myocardium and isolated ventricular myocytes demonstrated that this agent induced concentration-dependent translocation of the delta-isoform but not the other isoforms of PKC to the plasma membrane. CONCLUSIONS: The mechanism of cardioprotection by JTV519 against ischemia/reperfusion injury involves isozyme-specific PKC activation through a receptor-independent mechanism. This agent may provide a novel pharmacological approach for the treatment of patients with acute coronary diseases via a subcellular mechanism mimicking ischemic preconditioning.

Mast cells cause apoptosis of cardiomyocytes and proliferation of other intramyocardial cells in vitro.

BACKGROUND: Mast cells are multifunctional cells containing various mediators such as cytokines, proteases, and histamine. They are found in the human heart and have been implicated in ventricular hypertrophy and heart failure. However, their roles in pathogenesis of these diseases are unknown. METHODS AND RESULTS: Cultured cardiomyocytes from neonatal rats were incubated with mast cell granules (MCGs) for 24 hours. The highest concentration of diluted MCGs caused the death of approximately 70% of cardiomyocytes. This cell death was proved to be apoptosis, as quantified by electron microscopy and biochemical criteria. MCG-mediated cytotoxicity was prevented by pretreatment of MCGs with protease inhibitors or a neutralizing antibody against rat mast cell chymase 1 (RMCP 1). RMCP 1 by itself was proved to induce cell death of cardiomyocytes. These results suggest that RMCP 1 contained in MCGs causes the death of cardiomyocytes. In contrast, MCGs induced the proliferation of intramyocardial cells other than myocytes. RMCP 1 was also proved to induce their proliferation. CONCLUSIONS: Mast cells cause apoptosis of cardiomyocytes and proliferation of other intramyocardial cells via the activity of RMCP 1. Our results suggest that mast cell chymase may play a role in the progression of heart failure, because loss of cardiomyocytes and proliferation of nonmyocardial cells exaggerate its pathophysiology.

Activation of lectin-like oxidized low-density lipoprotein receptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes.

BACKGROUND: Lectin-like oxidized LDL receptor-1 (LOX-1) was originally identified as a receptor expressed predominantly in endothelial cells. LOX-1 can also be expressed in other cell types, and the activation of the LOX-1 pathway has been implicated in apoptosis. There have been no reports, however, about LOX-1 expression in cardiac myocytes or regulation of myocardial cell apoptosis by LOX-1. METHODS AND RESULTS: In primary cardiac myocytes from neonatal rats, immunohistochemical analyses using a specific monoclonal antibody against LOX-1 demonstrated that LOX-1 expression was markedly induced by stimulation with norepinephrine and endothelin-1. LOX-1 expression was upregulated in cardiac myocytes as well as in vessel walls of failing rat hearts in vivo. In the presence of a low concentration of oxidized LDL that did not induce apoptosis by itself, artificial overexpression of LOX-1 in cardiac myocytes in culture resulted in apoptosis. LOX-1 overexpression induced activation of p38 mitogen-activated protein kinase (MAPK) and oxidative stress in cardiac myocytes, as demonstrated by an increase in positive immunostaining for 8-hydroxy-2'-deoxyguanosine. Inhibition of p38 MAPK by cotransfection of a dominant-negative form of MKK6 as well as by administration of a specific inhibitor, SB203580 or FR167653, almost completely blocked the induction of apoptosis by LOX-1 activation. Antioxidant catalase also blocked LOX-1-induced apoptosis as well as activation of p38 MAPK. CONCLUSIONS: These findings demonstrate that LOX-1 expression in cardiac myocytes is induced by neurohormonal factors activated in heart failure and that LOX-1-dependent apoptosis in these cells requires p38 MAPK, a component of oxidant stress-sensitive signaling pathways.