Effects of levosimendan on cardiac remodeling and cardiomyocyte apoptosis in hypertensive Dahl/Rapp rats.

BACKGROUND AND PURPOSE: Progression of heart failure in hypertensive Dahl rats is associated with cardiac remodeling and increased cardiomyocyte apoptosis. This study was conducted to study whether treatment with a novel inotropic vasodilator compound, levosimendan, could prevent hypertension-induced cardiac remodeling and cardiomyocyte apoptosis. EXPERIMENTAL APPROACH: 6-week-old salt-sensitive Dahl/Rapp rats received levosimendan (0.3 mg kg(-1) and 3 mg kg(-1) via drinking fluid) and high salt diet (NaCl 7%) for 7 weeks, Dahl/Rapp rats on low-salt diet served as controls. Blood pressure, cardiac functions by echocardiography, cardiomyocyte apoptosis by TUNEL technique, tissue morphology, myocardial expression of calcium cycling proteins, and markers of neurohumoral activation were determined. KEY RESULTS: Untreated Dahl/Rapp rats on high salt diet developed severe hypertension, cardiac hypertrophy and moderate systolic dysfunction. 38% of Dahl/Rapp rats (9/24) survived the 7-week-follow-up period. Cardiomyocyte apoptosis was increased by 6-fold during high salt diet. Levosimendan improved survival (survival rates in low- and high-dose levosimendan groups 12/12 and 9/12, p<0.001 and p=0.05, respectively), increased cardiac function, and ameliorated cardiac hypertrophy. Levosimendan dose-dependently prevented cardiomyocyte apoptosis. Levosimendan normalized salt-induced increased expression of natriuretic peptide, and decreased urinary noradrenaline excretion. Levosimendan also corrected salt-induced decreases in myocardial SERCA2a protein expression and myocardial SERCA2a/NCX-ratio. CONCLUSIONS AND IMPLICATIONS: Improved survival by the novel inotropic vasodilator levosimendan in hypertensive Dahl/Rapp rats is mediated, at least in part, by amelioration of hypertension-induced cardiac remodeling and cardiomyocyte apoptosis.

The control of cardiomyocyte apoptosis via the beta-adrenergic signaling pathways.

Cardiomyocyte death resulting from apoptosis has been implicated in the evolution of heart failure. In this review, we focus on the concept that the cardiotoxicity of excessive sympathetic nervous system activity observed in heart failure is in part due to myocytes death by apoptosis. In vitro, high doses of norepinephrine induce adult cardiomyocyte apoptosis via 3-adrenergic receptor-coupled signaling pathways (PKA and Ca2+ entry-dependent mechanisms). beta1-and beta2-AR co-exist in the cardiac cell. beta1-AR stimulation is pro-apoptotic, whereas beta2-AR stimulation is anti-apoptotic, mediating its protective effect via coupling to Gi. These in vitro observations have been confirmed in transgenic mice: cardiac beta1-AR overexpression increases apoptosis and leads to heart failure, whereas cardiac beta2-AR overexpression has no deleterious effects. beta-AR stimulation activates p38 kinases and JNK (via the small GTP protein Rac1); and exert anti- and pro-apoptotic effects, respectively. Other studies suggest that beta1-AR-stimulated apoptosis is dependent on Ca2+ -activated calmodulin kinase II and that the anti-apoptotic effect of beta2-AR is mediated via Akt-coupled pathways. beta-AR-stimulated apoptosis involves the mitochondrial pathway. Inhibition of mitochondrial permeability transition pore opening or caspase activation decreases beta-AR-stimulated apoptosis. Reactive oxygen species production is also involved in this process since superoxide dismutase/catalase-mimetics or catalase overexpression prevent beta-AR-stimulated apoptosis. In vivo, it has been shown that beta-AR blockers such as metoprolol and carvedilol have beneficial effects in animal models of chronic heart failure, associated with reduced apoptosis and improved cardiac systolic function. Understanding the mechanisms involved in the control of myocyte loss by the beta-adrenergic system will have direct clinical implications by improving the treatment of heart failure.

Adrenergic regulation of cardiac myocyte apoptosis.

The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of beta1-AR induces apoptosis which is cAMP-dependent and involves the voltage-dependent calcium influx channel. In contrast, stimulation of beta2-AR exerts an anti-apoptotic effect which appears to be mediated by a pertussis toxin-sensitive G protein. Stimulation of alpha1-AR causes myocyte hypertrophy and may exert an anti-apoptotic action. In transgenic mice, myocardial overexpression of either beta1-AR or G(alpha)s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of beta2-AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype alpha1B-AR does not result in apoptosis, whereas overexpression of G(alpha)q results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen-activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways.

Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes.

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ((3)H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an approximately 3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.

Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction.

Nitric oxide produced by inducible nitric oxide synthase (NOS2) has been implicated in the pathophysiology of chronic myocardial remodeling and failure. We tested the role of NOS2 in left ventricular (LV) remodeling early (1 month) and late (4 months) after myocardial infarction (MI) in mice lacking NOS2. MI size measured 7 days, 1 month, and 4 months after MI was the same in NOS2 knockout (KO) and wild-type (WT) mice. The LV end-diastolic pressure-volume relationship measured by the isovolumic Langendorff technique showed a progressive rightward shift from 1 to 4 months after MI in WT mice. LV developed pressure measured over a range of LV volumes was reduced at 1 and 4 months after MI in WT mice (P<0.05 and P<0.01 versus shams, respectively). In KO mice, the rightward shift was similar to that in WT mice at 1 and 4 months after MI, as was peak LV developed pressure at 1 month after MI. In contrast, at 4 months after MI, peak LV developed pressure in KO mice was higher than in WT mice (P<0.05 versus WT) and similar to that in sham-operated mice. At 1 month after MI, the frequency of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive myocytes in the remote myocardium was increased to a similar extent in WT and KO mice. At 4 months after MI, the frequency of apoptotic myocytes was increased in WT mice but not in KO mice (P<0.05 versus WT). Improved contractile function and reduced apoptosis were associated with reduced mortality rate in KO mice at 4 months after MI. Thus, NOS2 does not play an important role in determining infarct size or early LV remodeling during the first month after MI. In contrast, during late (ie, 4 months after MI) remodeling, NOS2 in remote myocardium contributes to decreased contractile function, increased myocyte apoptosis in remote myocardium, and reduced survival.

A randomized multicenter study comparing the efficacy and safety of intravenous milrinone and intravenous nitroglycerin in patients with advanced heart failure.

A randomized, open-label, parallel-group design was used to determine the percentage of patients achieving improvements in predetermined baseline hemodynamic end points (>20% to 30% increase in cardiac index depending on baseline values and >25% decrease in pulmonary capillary wedge pressure), assessed at hour 0 (end of initial dose titration) and 1, 2, 4, 8, and 24 hours after the infusion of milrinone or nitroglycerin. In total, 125 patients (60 milrinone, 65 nitroglycerin) enrolled in this study, and 119 (58 milrinone, 61 nitroglycerin) were evaluable for the efficacy analysis. A significantly greater proportion of milrinone-treated patients reached (45% v 14%, P =.005) and maintained (24% v 6%, P =.026) hemodynamic goals than did nitroglycerin-treated patients; the time to achieve hemodynamic goals was significantly less in milrinone-treated patients (33 +/- 2 v 54 +/- 10 minutes, P <.001). Milrinone was also significantly more effective in decreasing systemic vascular resistance (P =.004), increasing stroke volume (P =.008), and improving global clinical status. Inodilator therapy with milrinone seems more efficacious in attaining sustained hemodynamic improvement than does pure intravenous vasodilator therapy with nitroglycerin in treating patients with decompensated heart failure.

Influence of carvedilol on hospitalizations in heart failure: incidence, resource utilization and costs. U.S. Carvedilol Heart Failure Study Group.

BACKGROUND: Carvedilol reduces disease progression in heart failure, but to our knowledge, its effects on hospitalizations and costs have not been evaluated. OBJECTIVES: We examined the effects on hospitalization frequency and costs in the U.S. Carvedilol Heart Failure Trials Program. This program consisted of four concurrent, multicenter, double-blind, placebo-controlled studies involving 1,094 patients with New York Heart Association class II to IV heart failure, which treated patients with placebo or carvedilol for up to 15 months (median, 6.5 months). METHODS: Detailed resource utilization data were collected for all hospitalizations occurring between randomization and the end of follow-up. In-patient care costs were estimated based on observed levels of resource use. RESULTS: Compared with placebo, carvedilol reduced the risk of hospitalization for any reason by 29% (p = 0.009), cardiovascular hospitalizations by 28% (p = 0.034) and heart failure hospitalizations by 38% (p = 0.041). Carvedilol also decreased the mean number of hospitalizations per patient (for cardiovascular reasons 30% [p = 0.02], for heart failure 53% [p = 0.03]). Among hospitalized patients, carvedilol reduced severity of illness during hospital admission, as reflected by shorter length of stay and less frequent use of intensive care. For heart failure hospital admissions, carvedilol decreased mean length of stay by 37% (p = 0.03) and mean number of intensive care unit/coronary care unit days by 83% (p = 0.001), with similar effects on cardiovascular admissions. As a result, estimated inpatient care costs with carvedilol were 57% lower for cardiovascular admissions (p = 0.016) and 81% lower for heart failure admissions (p = 0.022). CONCLUSIONS: Carvedilol added to angiotensin-converting enzyme inhibition reduces hospitalization risk as well as severity of illness and resource utilization during admission in patients with chronic heart failure.

Race and the response to adrenergic blockade with carvedilol in patients with chronic heart failure.

BACKGROUND: The benefits of angiotensin-converting-enzyme inhibitors and beta-blockers may be smaller in black patients than in patients of other races, but it is unknown whether race influences the response to carvedilol in patients with chronic heart failure. METHODS: In the U.S. Carvedilol Heart Failure Trials Program, 217 black and 877 nonblack patients (in New York Heart Association class II, III, or IV and with a left ventricular ejection fraction of no more than 0.35) were randomly assigned to receive placebo or carvedilol (at doses of 6.25 to 50 mg twice daily) for up to 15 months. The effects of carvedilol on ejection fraction, clinical status, and major clinical events were retrospectively compared between black and nonblack patients. RESULTS: As compared with placebo, carvedilol lowered the risk of death from any cause or hospitalization for any reason by 48 percent in black patients and by 30 percent in nonblack patients. Carvedilol reduced the risk of worsening heart failure (heart failure leading to death, hospitalization, or a sustained increase in medication) by 54 percent in black patients and by 51 percent in nonblack patients. The ratios of the relative risks associated with carvedilol for these two outcome variables in black as compared with nonblack patients were 0.74 (95 percent confidence interval, 0.42 to 1.34) and 0.94 (95 percent confidence interval, 0.43 to 2.05), respectively. Carvedilol also improved functional class, ejection fraction, and the patients' and physicians' global assessments in both the black patients and the nonblack patients. For all these measures of outcome and clinical status, carvedilol was superior to placebo within each racial cohort (P<0.05 in all analyses), and there was no significant interaction between race and treatment (P> 0.05 in all analyses). CONCLUSIONS: The benefit of carvedilol was apparent and of similar magnitude in both black and nonblack patients with heart failure.

Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: role of p42/44 mitogen-activated protein kinase and reactive oxygen species.

Using spontaneously hypertensive and aortic banded rats, we have shown that expression of myocardial osteopontin, an extracellular matrix protein, coincides with the development of heart failure and is inhibited by captopril, suggesting a role for angiotensin II (ANG II). This study tested whether ANG II induces osteopontin expression in adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC), and if so, whether induction is mediated via activation of mitogen-activated protein kinases (p42/44 MAPK) and involves reactive oxygen species (ROS). ANG II (1 microM, 16 h) increased osteopontin expression (fold increase 3.3+/-0.34, n = 12, P < 0.01) in CMEC as measured by northern analysis, but not in ARVM. ANG II stimulated osteopontin expression in CMEC in a time- (within 4 h) and concentration-dependent manner, which was prevented by the AT1 receptor antagonist, losartan. ANG II elicited robust phosphorylation of p42/44 MAPK as measured using phospho-specific antibodies, and increased superoxide production as measured by cytochrome c reduction and lucigenin chemiluminescence assays. These effects were blocked by diphenylene iodonium (DPI), an inhibitor of the flavoprotein component of NAD(P)H oxidase. PD98059, an inhibitor of p42/44 MAPK pathway, and DPI each inhibited ANG II-stimulated osteopontin expression. Northern blot analysis showed basal expression of p22phox, a critical component of NADH/NADPH oxidase system, which was increased 40-60% by exposure to ANG II. These results suggest that p42/44 MAPK is a critical component of the ROS-sensitive signaling pathways activated by ANG II in CMEC and plays a key role in the regulation of osteopontin gene expression. Published 2001 Wiley-Liss, Inc.

Exaggerated left ventricular dilation and reduced collagen deposition after myocardial infarction in mice lacking osteopontin.

Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (P=NS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (P<0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive myocytes was similar in KO and WT mice after MI. In contrast, post-MI LV chamber dilation was approximately twice as great in KO versus WT mice (P<0.001). Myocyte length increased after MI in WT mice (P<0.001) but not in KO mice. Electron microscopy showed increased collagen content in WT mice after MI but not in KO mice after MI. Type I collagen content was increased approximately 3-fold and approximately 7-fold in remote and infarcted regions, respectively, of WT hearts after MI but not in KO hearts (P<0.01 versus WT hearts). Likewise, Northern analyses showed increased collagen I(alpha(1)) mRNA after MI in remote regions of WT hearts but not in KO hearts. Thus, increased OPN expression plays an important role in regulating post-MI LV remodeling, at least in part, by promoting collagen synthesis and accumulation.

Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. METHODS AND RESULTS: Experimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 10(6) myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of approximately 30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. CONCLUSIONS: Implanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

Nesiritide for the treatment of decompensated heart failure.

Nesiritide (human recombinant B-type natriuretic peptide) binds to receptors in the vasculature, kidney, and other organs to mimic the actions of endogenous natriuretic peptides. Intravenous infusion of nesiritide has been studied in more than 1,700 patients with acute decompensated heart failure (HF). Nesiritide causes potent, dose-related vasodilation that is rapid in onset and sustained for the duration of drug infusion. There is balanced arterial and venous dilation as reflected by decreases in systemic vascular resistance, systemic arterial pressure, pulmonary capillary wedge pressure, right atrial pressure, and mean pulmonary arterial pressure. Vasodilation occurs without a change in heart rate and is associated with increases in stroke volume and cardiac output. Nesiritide may promote diuresis because of a direct natriuretic action, increased cardiac output, and/or decreased aldosterone levels. In patients hospitalized for decompensated HF, nesiritide improves symptoms and is well tolerated. The major adverse effect is dose-related hypotension. Nesiritide is thus an attractive new vasodilator that should be valuable in the treatment of patients hospitalized for acute decompensated HF.

MEK1/2-ERK1/2 mediates alpha1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes.

We examined the relative roles of the mitogen-activated protein kinases (MAPK) in mediating the alpha1-adrenergic receptor (alpha1-AR) stimulated hypertrophic phenotype in adult rat ventricular myocytes (ARVM). Norepinephrine (NE; 1 microM) in the presence of the beta -AR antagonist propranolol (Pro; 2 microM) caused activation of Ras (>six-fold), MAPK/ERK kinase 1 and 2 (MEK1/2, >10-fold) and extracellular signal-regulated kinases 1 and 2 (ERK1/2, approximately 30-fold) within 5 min, as determined by kinase activity assays and Western blots using phospho-specific antibodies. Conversely, p38 and c-Jun amino-terminal kinases (JNK) were not activated by NE/Pro. Activated MEK1/2 signals remained detectable at 2 h, and activated ERK1/2 remained detectable at 48 h. The alpha1-AR selective inhibitor prazosin (100 nM) completely inhibited the NE/Pro-stimulated activation of Ras, MEK1/2 and ERK1/2. The MEK inhibitor PD98059 caused a concentration-dependent inhibition of NE/Pro-stimulated protein synthesis (as assessed by [3H]leucine incorporation and cellular protein accumulation) and ERK1/2 activation, with approximately 50% inhibition at a concentration between 10 and 50 microM, which is consistent with the known IC50 values of PD98059 for MEK1 (4 microM) and MEK2 (50 microM). Thus, these data show that alpha1-AR stimulated hypertrophy in ARVM is dependent on the MEK1/2-ERK1/2 signaling pathway.

ET(A)-receptor blockade prevents matrix metalloproteinase activation late postmyocardial infarction in the rat.

Endothelin (ET) A (ET(A)) receptors activate matrix metalloproteinases (MMP). Since endothelin-1 (ET) is increased in myocardium late postmyocardial infarction (MI), we hypothesized that stimulation of ET(A) receptors contributes to activation of myocardial MMPs late post-MI. Three days post-MI, rats were randomized to treatment with the ET(A)-selective receptor antagonist sitaxsentan (n = 12) or a control group (n = 12). Six weeks later, there were rightward shifts of the left ventricular (LV) end-diastolic and end-systolic pressure-volume relationships, as measured ex vivo by the isovolumic Langendorff technique. Both shifts were markedly attenuated by sitaxsentan. In LV myocardium remote from the infarct, the activities of MMP-1, MMP-2, and MMP-9 were increased in the post-MI group, and the increases were prevented by sitaxsentan treatment. Expression of tissue inhibitor of MMP-1 was decreased post-MI, and the decrease was prevented by sitaxsentan treatment. Chronic post-MI remodeling is associated with activation of MMPs in myocardium remote from the infarct. Inhibition of ET(A) receptors prevents MMP activation and LV dilation, suggesting that ET, acting via the ET(A) receptor, contributes to chronic post-MI remodeling by its effects on MMP activity.

Reactive oxygen species mediate alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes.

Norepinephrine (NE) causes hypertrophic growth of cardiac myocytes via stimulation of alpha1-adrenergic receptors (alpha1-AR). Reactive oxygen species (ROS) can act as signaling molecules for cell growth. Accordingly, we tested the hypothesis that ROS mediate alpha1-AR-stimulated hypertrophic growth in adult rat ventricular myocytes (ARVM). NE increased the level of intracellular ROS as assessed by lucigenin chemiluminescence or cytochrome c reduction, and this effect was prevented by the superoxide dismutase (SOD)-mimetic MnTMPyP. NE also caused the induction of MnSOD mRNA. alpha1-AR stimulation with NE (1 microM) in the presence of propranolol (2 microM) for 48-96 h caused a hypertrophic growth phenotype characterized by a 36+/-3% increase in 3H-leucine incorporation, a 49+/-14% increase in protein accumulation, a six-fold induction of atrial natriuretic peptide mRNA, actin filament reorganization, and the induction of MnSOD mRNA. These responses were all prevented by pretreatment with the alpha1-AR-selective antagonist prazosin (100 n M) or the SOD-mimetics MnTMPyP (50 microM) and Euk-8 (100 microM). MnTMPyP had no effect on alpha1-AR-stimulated 3H-inositol phosphate turnover or the hypertrophic phenotype caused by the protein kinase C activator phorbol-12-myristate-13-acetate. Thus, ROS play a critical role in mediating the hypertrophic growth response to alpha1-AR-stimulation in ARVM.

Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts.

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H(2)O(2) (0.05-5 microM) or the superoxide-generating system xanthine (500 microM) plus xanthine oxidase (0.001-0.1 mU/ml) (XXO) for 24 h. In-gel zymography demonstrated that H(2)O(2) and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-13, MMP-2, and MMP-9. H(2)O(2) and XXO decreased collagen synthesis (collagenase-sensitive [(3)H]proline incorporation) without affecting total protein synthesis ([(3)H]leucine incorporation). H(2)O(2) and XXO decreased the expression of procollagen alpha(1)(I), alpha(2)(I), and alpha(1)(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H(2)O(2), but not XXO, also decreased the expression of nonfibrillar procollagen alpha(1)(IV) and alpha(2)(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DDC, 100 microM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone 3-acetate (DHEA; 10 microM) to increase intracellular H(2)O(2). DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an SOD/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.

Survival and prognosis: investigation of Crataegus extract WS 1442 in congestive heart failure (SPICE)--rationale, study design and study protocol.

UNLABELLED: SPICE is the first, international, randomized, placebo-controlled, double-blind study to investigate the influence of the herbal drug Crataegus Special Extract WS 1442 (hawthorn leaves with flowers) on mortality of patients suffering from congestive heart failure. BACKGROUND: In vitro and experimental animal studies have suggested the following pharmacological modes of action of standardized Crataegus extracts: (1) cAMP-independent positive inotropy; (2) peripheral and coronary vasodilation; (3) protection against ischemia-induced ventricular arrhythmias; (4) antioxidative properties; and (5) anti-inflammatory effects. STUDY DESIGN: In this randomized, placebo-controlled, double-blind, international trial (approximately 120 investigational centers in seven European countries), up to 2300 patients with congestive heart failure, New York Heart Association class II and III and markedly impaired left ventricular function, will be enrolled and treated over a period of 24 months. During this time patients receive either two film-coated tablets of 450 mg of the Special Extract WS 1442 standardized to 84.3 mg of oligomeric procyanidines or matched placebo per day in addition to standard therapy for congestive heart failure, such as diuretics, digoxin or digitoxin, beta-adrenoceptor blockers and angiotensin-converting-enzyme inhibitors. The primary outcome variable is the combined endpoint of cardiac death, non-lethal myocardial infarction, and hospitalization due to progression of heart failure. Secondary outcome variables are total mortality, exercise duration, echocardiographic parameters, quality of life as well as pharmacoeconomic parameters. The first patient was included in October 1998. The trial is expected to be completed at the end of 2002.