Bosentan enhances viral load via endothelin-1 receptor type-A-mediated p38 mitogen-activated protein kinase activation while improving cardiac function during coxsackievirus-induced myocarditis.

Reduced cardiac output is one of the consequences of myocarditis. Bosentan, an endothelin-1 receptor (ET1R) antagonist, could be useful to reduce cardiac afterload, preserving cardiac output. In this study, we investigated the potential therapeutic use of bosentan in an animal model of viral myocarditis. Using a mouse model of coxsackievirus B3 (CVB3)-induced myocarditis, we demonstrated preserved ejection fraction (EF) and fractional shortening (FS) by treatment with bosentan (68+/-5.8% EF and 40+/-3.7% FS for treated versus 48+/-2.2% EF and 25+/-2.6% FS for controls; P=0.028). However, bosentan enhanced cardiac viral load (10.4+/-6.7% in the bosentan group versus 5.0+/-5.5% in control group; P=0.02), likely through enhancement of p38 mitogen-activated protein kinase (MAPK) phosphorylation (0.77+/-0.40% ATF2 activation in the bosentan group versus 0.03+/-0.02% in controls; P=0.0002), mediated by endothelin receptor type-A. We further demonstrate that a water soluble inhibitor of p38 MAPK, SB203580 HCl, is a potent inhibitor of virus replication in the heart (0.28% antisense viral genome stained area for 3 mg/kg dose versus 2.9% stained area for controls; P=0.01), attenuates CVB3-induced myocardial damage (blinded cardiac histopathologic scores of 1.8+/-1.6 and 2.05+/-1.2 for the 3 mg/kg and 10 mg/kg doses, respectively, versus 3.25+/-1.2 for the controls), and preserves cardiac function (69+/-3.5% EF for 3 mg/kg dose and 71+/-6.7% EF for 10 mg/kg dose versus 60+/-1.5% EF control; P=0.038 and P=0.045, as compared to control, respectively). Bosentan, a prescribed vasodilator, improves cardiac function but enhances viral load and myocarditis severity through ETRA mediated p38 MAPK activation; p38 MAPK is a desirable antiviral target. Caution must be exercised during treatment of suspected infectious myocarditis with supportive vasoactive remedies.

A cardiac myosin light chain kinase regulates sarcomere assembly in the vertebrate heart.

Marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing human myocardium. Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v), which is involved in the development of human cardiomyopathy, is an important structural protein that affects physiologic cardiac sarcomere formation and heart development. Integrated cDNA expression analysis of failing human myocardia uncovered a novel protein kinase, cardiac-specific myosin light chain kinase (cardiac-MLCK), which acts on MLC2v. Expression levels of cardiac-MLCK were well correlated with the pulmonary arterial pressure of patients with heart failure. In cultured cardiomyocytes, knockdown of cardiac-MLCK by specific siRNAs decreased MLC2v phosphorylation and impaired epinephrine-induced activation of sarcomere reassembly. To further clarify the physiologic roles of cardiac-MLCK in vivo, we cloned the zebrafish ortholog z-cardiac-MLCK. Knockdown of z-cardiac-MLCK expression using morpholino antisense oligonucleotides resulted in dilated cardiac ventricles and immature sarcomere structures. These results suggest a significant role for cardiac-MLCK in cardiogenesis.

Neuronal angiotensin II type 1 receptor upregulation in heart failure: activation of activator protein 1 and Jun N-terminal kinase.

Chronic heart failure (CHF) is a leading cause of mortality in developed countries. Angiotensin II (Ang II) plays an important role in the development and progression of CHF. Many of the important functions of Ang II are mediated by the Ang II type 1 receptor (AT(1)R), including the increase in sympathetic nerve activity in CHF. However, the central regulation of the AT(1)R in the setting of CHF is not well understood. This study investigated the AT(1)R in the rostral ventrolateral medulla (RVLM) of rabbits with CHF, its downstream pathway, and its gene regulation by the transcription factor activator protein 1 (AP-1). Studies were performed in 5 groups of rabbits: sham (n=5), pacing-induced (3 to 4 weeks) CHF (n=5), CHF with intracerebroventricular (ICV) losartan treatment (n=5), normal with ICV Ang II treatment (n=5), and normal with ICV Ang II plus losartan treatment (n=5). AT(1)R mRNA and protein expressions, plasma Ang II, and AP-1-DNA binding activity were significantly higher in RVLM of CHF compared with Sham rabbits (240.4+/-30.2%, P<0.01; 206.6+/-25.8%, P<0.01; 280+/-36.5%, P<0.05; 207+/-16.4%, P<0.01, respectively). Analysis of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) pathway showed that phosphorylated c-Jun proteins, phosphorylated JNK proteins, and JNK activity increased significantly in RVLM of CHF compared with sham (262.9+/-48.1%, 213.8+/-27.7%, 148.2+/-10.1% of control, respectively). Importantly, ICV losartan in CHF rabbits attenuated these increases. ICV Ang II in normal rabbits simulated the molecular changes seen in CHF. This effect was blocked by concomitant ICV losartan. In addition, Ang II-induced AT(1)R expression was blocked by losartan and a JNK inhibitor, but not by extracellular signal-regulated kinase or p38 MAP kinase inhibitors in a neuronal cell culture. These data suggest that central Ang II activates the AT(1)R, SAPK/JNK pathway. AP-1 may further regulate gene expression in RVLM in the CHF state.

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function.

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.

Altered creatine kinase adenosine triphosphate kinetics in failing hypertrophied human myocardium.

BACKGROUND: The progression of pressure-overload left ventricular hypertrophy (LVH) to chronic heart failure (CHF) may involve a relative deficit in energy supply and/or delivery. METHODS AND RESULTS: We measured myocardial creatine kinase (CK) metabolite concentrations and adenosine triphosphate (ATP) synthesis through CK, the primary energy reserve of the heart, to test the hypothesis that ATP flux through CK is impaired in patients with LVH and CHF. Myocardial ATP levels were normal, but creatine phosphate levels were 35% lower in LVH patients (n = 10) than in normal subjects (n = 14, P < 0.006). Left ventricular mass and CK metabolite levels in LVH were not different from those in patients with LVH and heart failure (LVH+CHF, n = 10); however, the myocardial CK pseudo first-order rate constant was normal in LVH (0.36 +/- 0.04 s(-1) in LVH versus 0.32 +/- 0.06 s(-1) in normal subjects) but halved in LVH+CHF (0.17 +/- 0.06 s(-1), P < 0.001). The net ATP flux through CK was significantly reduced by 30% in LVH (2.2 +/- 0.7 micromol x g(-1) x s(-1), P = 0.011) and by a dramatic 65% in LVH+CHF (1.1 +/- 0.4 micromol x g(-1) x s(-1), P < 0.001) compared with normal subjects (3.1 +/- 0.8 micromol x g(-1) x s(-1)). CONCLUSIONS: These first observations in human LVH demonstrate that it is not the relative or absolute CK metabolite pool sizes but rather the kinetics of ATP turnover through CK that distinguish failing from nonfailing hypertrophic hearts. Moreover, the deficit in ATP kinetics is similar in systolic and nonsystolic heart failure and is not related to the severity of hypertrophy but to the presence of CHF. Because CK temporally buffers ATP, these observations support the hypothesis that a deficit in myofibrillar energy delivery contributes to CHF pathophysiology in human LVH.

Improvement of peripheral endothelial dysfunction by protein tyrosine phosphatase inhibitors in heart failure.

BACKGROUND: Chronic heart failure (CHF) induces endothelial dysfunction characterized by a decrease in nitric oxide (NO) production in response to flow (flow-mediated dilatation [FMD]). Because activation of endothelial NO synthase (eNOS) by flow requires tyrosine phosphorylation, we tested whether endothelial dysfunction could be corrected by increasing phosphotyrosine levels using protein tyrosine phosphatase (PTP) inhibitors and especially inhibitors of PTP1B. METHODS AND RESULTS: CHF was induced by coronary ligation in mice, and FMD was assessed in isolated and cannulated mesenteric artery segments (2 mm in length and <300 microm in diameter). CHF almost abolished FMD but only moderately affected the response to acetylcholine. In mice with CHF, the PTP1B inhibitors AS279, AS098, and AS713 restored FMD to levels similar to those of normal mice. This restoration was reduced by inhibitors of eNOS and phosphatidylinositol-3 kinase. Polymerase chain reaction and Western blot showed that arteries express PTP1B, and this expression was not affected by CHF. Immunolocalization revealed the presence of PTP1B in the endothelium and the adventitia. Flow induced a transient eNOS phosphorylation that was absent in CHF. PTP1B inhibition stimulated early eNOS phosphorylation and increased phosphorylation of Akt. CONCLUSIONS: Our results demonstrate for the first time that PTP1B inhibitors may be potent treatments for endothelial dysfunction.

Myocardial extracellular matrix remodeling in ischemic heart failure.

Left ventricular (LV) remodeling is a process whereby structural alterations attempt to compensate altered hemodynamic load. In the chronic setting this process becomes maladaptive, self-sustaining and is associated with worsened survival. The extracellular matrix (ECM) of the heart, once believed an inert scaffold for cardiomyocytes, is now known to play an important role in LV remodeling. The enzyme system primarily responsible for ECM turnover is the matrix metalloproteinases (MMPs), and these enzymes are robustly altered in cardiovascular pathologies, including myocardial infarction (MI) and ischemic heart failure. A cause-and-effect relationship has been established between MMPs and LV remodeling post MI, as MMP inhibition prevents LV dilation and preserves cardiac function in animal models of infarction. In spite of this, initial clinical experience with MMP inhibition post MI has been disappointing. This review examines the structural and functional roles of the myocardial ECM, the evidence for MMP involvement in LV remodeling, and recent investigations into MMPs as prognostic markers and therapeutic targets.

Matrix metalloprotease activity is enhanced in the compensated but not in the decompensated phase of pressure overload hypertrophy.

BACKGROUND: During the transition of pressure overload hypertrophy (POH) to heart failure (HF) there is intense interstitial cardiac remodeling, characterized by a complex balance between collagen deposition and degradation by matrix metalloproteases (MMPs). This study was aimed at investigating the process of cardiac remodeling during the different phases of the transition of POH to HF. METHODS: Guinea pigs underwent thoracic descending aortic banding or sham operation. Twelve weeks after surgery, left-ventricular (LV) end-diastolic internal dimension and ventricular systolic pressure were measured by combined M-mode echocardiography and micromanometer cathetherization. The MMP activity, tissue-specific MMP inhibitors (TIMPs), and collagen fraction were evaluated in LV tissue samples by zymography, ELISA, and computer-aided analysis, respectively. RESULTS: Banded animals were divided by lung weight values into either compensated left-ventricular hypertrophy (LVH) or HF groups, as compared with sham-operated controls. All HF animals exhibited a restrictive pattern of Doppler transmitral inflow, indicative of diastolic dysfunction, and developed lung congestion. Compensated LVH was associated with increased MMP-2 activity, which was blunted after transition to HF, at a time when TIMP-2 levels and collagen deposition were increased. CONCLUSIONS: The cardiac remodeling process that accompanies the development of POH is a phase-dependent process associated with progressive deterioration of cardiac function.

Level of beta-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure.

BACKGROUND: Heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased level of myocardial betaAR kinase 1 (betaARK1). Our previous studies have shown that inhibition of betaARK1 with the use of the Gbetagamma sequestering peptide of betaARK1 (betaARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of betaARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of betaARK1 inhibition correlates with the degree of heart failure. METHODS AND RESULTS: Transgenic (TG) mice with varying degrees of cardiac-specific expression of betaARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial betaARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of betaARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low betaARKct expression developed severe heart failure, whereas mice with high betaARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of betaARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal betaAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in betaAR function, similar to the WT mice. CONCLUSIONS: These data show that the level of betaARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when betaARK1 inhibition is considered as a molecular target.

Increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme Homologue ACE2.

BACKGROUND: The formation of angiotensin-(1-7) from either angiotensin (Ang) I or Ang II in failing human hearts is not well understood. METHODS AND RESULTS: Angiotensinase activity in left and right ventricular membranes from 14 idiopathic dilated cardiomyopathy (IDC), 8 primary pulmonary hypertension (PPH), and 13 nonfailing human hearts was measured with either 125I-Ang I or 125I-Ang II as substrate. Ang-(1-7)-forming activity from 125I-Ang I was inhibited by thiorphan. With 125I-Ang II as substrate, Ang-(1-7) formation was inhibited by the ACE2-specific inhibitor C16. Western blotting with an anti-ACE2 antibody confirmed the presence of ACE2. Angiotensinase activity with 125I-Ang I as substrate was increased in failing IDC left ventricles (LVs) compared with nonfailing LVs (P<0.001). Ang-(1-7)-forming activity with 125I-Ang II as substrate was increased in both failing LVs and right ventricles (RVs) of IDC hearts and only in failing RVs of PPH hearts (PPH LV, 51.12+/-5.25; PPH RV, 89.97+/-11.21; IDC LV, 139.7+/-21.96; and IDC RV, 192.7+/-5.43; NF LV, 32.89+/-5.38; NF RV 40.49+/-10.66 fmol/min per milligram (P<0.05 PPH RV versus PPH LV; P<0.05 PPH RV versus NF RV; P<0.001 IDC LV versus NF LV; P<0.001 IDC RV versus NF RV). CONCLUSIONS: Ang-(1-7)-forming activity from both Ang I and Ang II was increased in failing human heart ventricles but was mediated by at least two different angiotensinases. The first, which demonstrated substrate preference for Ang I, was neutral endopeptidase (NEP)-like. The second was ACE2, as demonstrated by Western blotting and inhibition of activity with C16.

Myocyte nitric oxide synthase 2 contributes to blunted beta-adrenergic response in failing human hearts by decreasing Ca2+ transients.

BACKGROUND: Human heart failure (HF) usually exhibits blunted response to beta-adrenergic receptor (AR) stimulation. Here, we examined whether expression of nitric oxide synthase-2 (NOS2, or inducible NOS) contributes to this loss of inotropic reserve in human HF. METHODS AND RESULTS: Failing human hearts were obtained at transplantation. Contraction and [Ca2+]i measurements were performed in isolated cardiac myocytes and trabeculae. In HF myocytes and muscle, isoproterenol (ISO), a beta-AR agonist, led to small inotropic and lusitropic responses. Specific inhibition of NOS2 by aminoguanidine (AG) or L-NIL dramatically increased the ISO-induced inotropy and lusitropy, such that the ISO+AG response in HF approached that seen with ISO alone in nonfailing human myocytes or muscles. Ca2+ transient data directly paralleled these results, indicating that altered cellular Ca2+ handling is responsible. In nonfailing human hearts, NOS2 inhibition had no effects. In addition, NOS2 inhibition also had no effect in 30% of failing hearts, but in these myocytes and muscles, the ISO response alone was similar to that of nonfailing hearts. In line with these functional findings, NOS2 protein expression measured by Western blotting was induced in HF when AG/L-NIL had a functional effect but not when AG/L-NIL had no effect on contractility and Ca2+ transients. CONCLUSIONS: NOS2 expression strongly limited ISO-induced increases in contraction, twitch Delta[Ca2+]i, and lusitropy in trabeculae and isolated myocytes from failing human hearts. Thus, the beta-AR hyporesponsiveness in human HF is mediated in large part by NO (or related congeners) produced within cardiac myocytes via NOS2.

Oxygen free radical release in human failing myocardium is associated with increased activity of rac1-GTPase and represents a target for statin treatment.

BACKGROUND: Reactive oxygen species (ROS) contribute to the development of heart failure. A potential source of myocardial ROS is the NADPH oxidase, which is regulated by the small GTP-binding protein rac1. Isoprenylation of rac1 can be inhibited by statin therapy. Thus, we examined ROS and rac1 in human failing myocardium and tested their regulation by statins in vivo. METHODS AND RESULTS: In human left ventricular myocardium from patients with ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM), NADPH oxidase activity was increased 1.5-fold compared with nonfailing controls (P<0.05, n=8). In failing myocardium, increased oxidative stress determined by measurements of lipid peroxidation and aconitase activity was associated with increased translocation of rac1 from the cytosol to the membrane. Pull-down assays revealed a 3-fold increase of rac1-GTPase activity in ICM and DCM. In parallel, membrane expression of the NADPH oxidase subunit p47phox, but not p67phox, was upregulated in failing compared with nonfailing myocardium. In right atrial myocardium from patients undergoing cardiac surgery who were prospectively treated with atorvastatin or pravastatin (40 mg/d, 4 weeks), rac1-GTPase activity was decreased to 67.9+/-12% and 65.6+/-13.8% compared with patients without statin (P<0.05, n=8). Both atorvastatin and pravastatin significantly reduced angiotensin II-stimulated but not basal NADPH oxidase activity. CONCLUSIONS: Failing myocardium of patients with DCM and ICM is characterized by upregulation of NADPH oxidase-mediated ROS release associated with increased rac1 activity. Oral statin treatment inhibits myocardial rac1-GTPase activity. These data suggest that extrahepatic effects of statins can be observed in humans and may be beneficial for patients with chronic heart failure.

Calcium/calmodulin-dependent protein kinase II activity is increased in sarcoplasmic reticulum from coronary artery ligated rabbit hearts.

A protein kinase activity intrinsic to the sarcoplasmic reticulum was studied in normal and hypertrophied rabbit hearts. The relationship between this kinase activity and phospholamban phosphorylation was examined. Calmodulin-dependent kinase II activity was found to be increased in sarcoplasmic reticulum preparations from hypertrophied hearts compared with normal. This was evident by measuring the phosphotransferase activity of the kinase and also by examining phospholamban phosphorylation by electrophoretic band shift analysis. Increased phospholamban phosphorylation by Calmodulin-dependent protein kinase II was dependent on prior phosphorylation by cAMP-dependent protein kinase, indicating potential crosstalk. Specific immunoblot analysis of the rabbit sarcoplasmic reticulum identified the presence of the delta form of calmodulin dependent protein kinase II and showed it to be up-regulated in hypertrophied hearts.

Coordinated shifts in Na/K-ATPase isoforms and their endogenous ligands during cardiac hypertrophy and failure in NaCl-sensitive hypertension.

OBJECTIVES: NaCl loading of Dahl salt-sensitive rats (DS) stimulates marinobufagenin (MBG), an alpha1 Na/K-ATPase (NKA) isoform ligand. Cardiac function depends on NKA, which is regulated in part by endogenous digitalis-like ligands. Our goal was to study whether changes occur in MBG and endogenous ouabain (EO) production during cardiac remodelling in hypertensive DS, and whether these are associated with changes in myocardial NKA isoforms and sensitivity to MBG and ouabain. METHODS: Changes in MBG and EO levels, changes in myocardial NKA isoform composition, and sensitivity to endogenous ligands during development of cardiac hypertrophy and the transition to heart failure were studied in DS rats with an 8% NaCl intake. RESULTS: The animals developed compensated left ventricular hypertrophy after 4 weeks, which progressed to heart failure at 9-12 weeks. The hypertrophic stage was associated with increased plasma MBG levels (mean +/- SEM of 1.22 +/- 0.22 versus 0.31 +/- 0.03 nmol/l; P < 0.01), increased sensitivity of NKA to MBG, and an increased abundance of alpha1 NKA. Plasma levels of EO did not change, and the sensitivity of NKA to ouabain decreased. The transition to heart failure was accompanied by a decrease in alpha1 NKA, a reduction in plasma MBG, and decreased sensitivity of NKA to MBG. In addition, an increased abundance of ouabain-sensitive alpha3 NKA, a three-fold rise in plasma EO (1.01 +/- 0.13 versus 0.27 +/- 0.06 nmol/l), and a seven-fold increase in the ouabain sensitivity of NKA compared with controls were observed. CONCLUSIONS: During cardiac hypertrophy and the transition to heart failure, a shift in endogenous NKA ligands production is linked to a shift in myocardial NKA isoforms.

Association of left ventricular systolic performance and cavity size with angiotensin-converting enzyme genotype in idiopathic dilated cardiomyopathy.

The insertion-deletion (ID) polymorphism of the angiotensin-converting enzyme (ACE) gene is a marker linked to differences in plasma and cardiac ACE activity as well as to an increased mortality in patients with idiopathic heart failure. We examined the possibility that ACE gene ID variants are associated with differences in left ventricular (LV) systolic performance or internal LV dimensions in a high-risk cohort of patients with idiopathic dilated cardiomyopathy (IDC). The ACE genotype was determined in 171 patients selected with IDC in New York Heart Association functional class II to III heart failure and with a LV ejection fraction of < or = 40%. Left ventricular performance and dimensions were assessed using echocardiography (n = 161) and radionuclide ventriculography (n = 169). The frequency of ACE gene ID alleles was not different in the study versus non-age-matched (n = 171; odds ratio 0.94) and age-matched (n = 106, odds ratio 0.88) control groups. Ejection fraction was found to be worse in patients with the DD genotype (echocardiography, DD = 23.5 +/- 0.70, ID + II = 26.8 +/- 0.8, p = 0.009; ventriculography, DD = 21.7 +/- 0.9, ID + II = 25.3 +/- 0.8, p = 0.003). LV end-systolic and end-diastolic diameters were increased in patients with the DD genotype. Multifactor regression analysis showed the ACE genotype to be an independent predictor of both ejection fraction (echocardiography, p <0.02; ventriculography, p <0.03) and end-diastolic diameter (p <0.02). In conclusion, the results of this study indicate that the DD genotype of the ACE gene is independently associated with both a reduced LV systolic performance and an increased LV cavity size in patients with IDC.

Delayed circulatory failure due to the induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in anaesthetized rats.

1. This study investigates the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aureus, a micro-organism without endotoxin, on haemodynamics and induction of nitric oxide synthase (iNOS) in the anaesthetized rat. 2. Intravenous injection of LTA (10 mg kg-1) resulted in a decrease in blood pressure from 123 +/- 1 mmHg to 83 +/- 7 mmHg after 270 min (P < 0.001) and a reduction of the pressor response to noradrenaline (1 microgram kg-1) from 33 +/- 1 mmHg.min to 23 +/- 3 mmHg.min after 270 min (P < 0.05). 3. The delayed circulatory failure (hypotension and vascular hyporeactivity) caused by LTA was prevented by pretreatment of rats with dexamethasone (10 mg kg-1, 60 min prior to LTA) or the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mg kg-1 h-1, i.v. infusion starting 30 min prior to LTA). 4. In contrast, treatment of rats with polymyxin B (0.05 mg kg-1), an agent which binds endotoxin (lipopolysaccharides, LPS), did not affect the delayed circulatory failure caused by LTA. Polymyxin B, however, attenuated the hypotension and vascular hyporeactivity to noradrenaline afforded by endotoxaemia (2 mg kg-1 LPS, i.v.) for 270 min. 5. The delayed circulatory failure caused by LTA was associated with a time-dependent increase in (i) the expression of iNOS protein in the lung (Western blot analysis), and (ii) iNOS activity. This increase in iNOS protein and activity was prevented by pretreatment of LTA-rats with dexamethasone (10 mg kg-1). 6. Intravenous injection of LTA resulted in an increase in serum tumour necrosis factor (TNF)-alpha(maximum at 90 min after LTA), which was attenuated by pretreatment of rats with dexamethasone(10 mg kg-1, 60 min prior to LTA). The magnitude of the rise in TNF-alpha caused by LTA was similar to the one elicited by LPS (10mgkg-', i.v.).7. Thus, an enhanced formation of nitric oxide following the induction of iNOS contributes importantly to the delayed vascular failure (hypotension and vascular hyporeactivity) caused by LTA in the anaesthetized rat. We suggest that the endogenous release of TNF-alpha contributes to the induction ofiNOS caused by LTA in vivo.

Altered number of diaphorase (NOS) positive neurons in the hypothalamus of rats with heart failure.

Recently, we have demonstrated a decreased neuronal isoform of nitric oxide synthase (nNOS) message in the hypothalamus of rats with heart failure (HF). The purpose of this study was to determine the changes in NADPH-diaphorase (a commonly used marker for neuronal NOS activity) positive neurons in specific hypothalamic sites of rats with HF. After a standard histochemical protocol, NOS positive neurons were measured in paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MePO), subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and lateral hypothalamus (LH) of rats with coronary artery ligation (HF group; n=8) and sham-operated control rats (n=9). A total of 4 months after coronary ligation, the rats in the HF group displayed infarcts greater than at least 35% of the left ventricular wall (n = 8). Sham-operated rats had no observable damage to the myocardium. Rats with HF had a significantly lower number of NOS positive cells in the PVN (36% less) compared to sham rats. The number of NOS positive cells remained unaltered in the SON, MePO and LH in rats with HF. Conversely there was an increased number of NOS positive cells in the SFO (42% greater) and OVLT (100% greater). These data support the conclusion that the NO system within the hypothalamus involved in controlling autonomic outflow is altered during HF and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in HF.

Cardiac effects of R 79595 and its isomers (R 80122 and R 80123) in an acute heart failure model. A new class of cardiotonic agents with highly selective phosphodiesterase III inhibitory properties.

R 79595 (N-cyclohexyl-N-methyl-2-[[[phenyl (1,2,3,5-tetrahydro-2 oxoimidazo [2,1-b]-quinazolin-7-yl) methylene] amin] oxy] acetamide) and its isomers represent a novel class of compounds with phosphodiesterase (PDE) inhibitory and cardiotonic (positive inotropic) actions. The cardiac effects of this class of compounds were investigated in the hexobarbital-depressed heart-lung preparation of the guinea-pig. After induction of heart failure (reduction of cardiac output to 25% of the initial value) cumulative addition of R 79595 or its isomers R 80122 (E-isomer) and R 80123 (Z-isomer) concentration-dependently reversed the cardiac depressant effects of hexobarbitone-Na. With regard to reconstitution of contractility and cardiac function R 80122 (E-isomer) was 10 fold more potent than R 79595 (1:1 mixture of the isomers) and nearly 100 fold more potent than R 80123 (Z-isomer). Furthermore, the cardiotonic action of the most potent isomer (R 80122) was compared to the effects of several positive inotropic reference compounds. The order of cardiotonic potency was as follows: (-)-adrenaline > R 80122 = adibendan > digitoxin > milrinone = enoximone > theophylline. Adibendan (EC50 value: 6.7 +/- 1.8 x 10.-8 mol/l), which showed cardiotonic effects in the same concentration range as R 80122 (EC50 value: 6.1 +/- 1.3 x 10(-8) mol/l), was significantly (p < 0.01) less effective than R 80122 with respect to the maximally induceable increase in cardiac output (CO). The cardiotonic effects of R 80122 could be observed in the low concentration range of 3 x 10(-8) to 1 x 10(-6) mol/l, whereas enoximone (EC50 value: 1.2 +/- 0.1 x 10(-5) mol/l) and milrinone (EC50 value: 8.9 +/- 3.5 x 10(-6) mol/l) elicited positive inotropic effects at 100 fold higher concentrations. Digitoxin was 10 fold less and theophylline was 300 fold less potent than R 80122 with regard to reconstitution of heart function. The cardiotonic effects of R 80122 were not accompanied by an increase in heart rate as found with milrinone, theophylline or (-)-adrenaline in this model. Furthermore, the PDE inhibitory effect of R 79595 and its E-isomer R 80122 were investigated in partially purified isoenzymes from guinea-pig ventricles. The IC50 values of R 79595 and R 80122 on PDE I-IV were compared to the IC50 values of adibendan, milrinone, enoximone and theophylline. The selectivity of an inhibitor for PDE III was evaluated by division of its IC50 values on PDE I, II and IV by the IC50 value on PDE III. R 80122 was the most potent and selective PDE III inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of saterinone and its enantiomers R(+)-saterinone and S(-)-saterinone on the phosphodiesterase isoenzymes from ventricular tissue of failing human hearts and porcine hearts.

Stereoisomers often exhibit differences in their pharmacological activities. Therefore, the phosphodiesterase inhibitory effects of the cardiotonic agent saterinone in form of the racemate were investigated in comparison with the inhibitory properties of its enantiomers R(+)- and S(-)-saterinone. For this purpose the phosphodiesterase isoenzymes from ventricular tissue of failing human hearts and porcine hearts were separated by DEAE-sepharose anion exchange chromatography. Four different phosphodiesterase isoenzymes were isolated from failing human myocardium and designated phosphodiesterase I-IV. Three phosphodiesterase isoenzymes could be separated from ventricular tissue of porcine hearts. A Ca2+/calmodulin stimulated phosphodiesterase I was not detectable in porcine myocardium. In failing human hearts racemic saterinone was a potent inhibitor of phosphodiesterase III (IC50 0.02 mumol/l) and IV (IC50 0.03 mumol/l) as compared to the inhibition of phosphodiesterase I (IC50 37.3 mumol/l) and II (IC50 51.4 mumol/l). In comparison with the racemate, R(+)- and S(-)-saterinone showed only slight differences in their phosphodiesterase inhibitory effects. R(+)-saterinone inhibited phosphodiesterase III slightly but significantly more potently and selectively than did S(-)-saterinone. Compared to the inhibition of phosphodiesterase I and II both enantiomers were similarly potent and selective inhibitors of phosphodiesterase III and IV. Similar results were obtained in porcine hearts. It is concluded that the racemate saterinone and its enantiomers (R(+)- and S(-)-saterinone are virtually equipotent concerning the inhibition of phosphodiesterase isoenzymes isolated from failing human hearts or porcine ventricular tissue. The enantiomers of saterinone did not exhibit distinct stereoselectivity in their phosphodiesterase inhibitory effects.