Effects of calcium sensitizer OR-1986 on a cardiovascular mortality and myocardial remodelling in hypertensive Dahl/Rapp rats.

Calcium-sensitizing agents have been shown to improve cardiac function in patients suffering from acute decompensated heart failure, however, their long-term effects on cardiac remodeling and cardiovascular mortality are still largely unknown. In the present study we tested the hypothesis whether OR-1896, an active and long-lasting metabolite of calcium sensitizer levosimendan, prevents cardiovascular mortality and hypertension-induced myocardial remodelling in salt-sensitive Dahl/Rapp rats. OR-1896 was given orally to Dahl/Rapp SS rats on high-salt diet (NaCl 7% w/w) for 7 weeks at two different doses (0.5 and 0.05 mg/kg). OR-1896 prevented salt-induced cardiovascular mortality (survival rate 75 % in OR-1896 treated groups vs 38 % in untreated controls, p<0.01), ameliorated cardiac hypertrophy and improved systolic functions of the heart without major influence on systemic blood pressure. OR-1896 also ameliorated salt-induced increase in cardiac ANP mRNA expression and plasma BNP level. Salt-induced cardiac remodelling was associated with 4-fold increase in cardiac p16(INK4a) mRNA expression, a marker of cellular senescence. OR-1896 dose-dependently ameliorated cardiomyocyte senescence. Our findings suggest a therapeutic role for OR-1896 in the prevention of cardiac remodelling in salt-sensitive forms of hypertension. The present study also underscores the importance of cellular senescence in the pathogenesis of salt-induced hypertensive heart disease.

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.

Improved survival with simendan after experimental myocardial infarction in rats.

This study compared the effects of simendan, a calcium sensitizer, with those of milrinone and enalapril on survival of rats with healed myocardial infarction. Seven days after ligation-induced myocardial infarction, the rats were randomized to control, milrinone, enalapril, or simendan groups. All compounds were administered via the drinking water for 312 days, at which time there was 80% mortality in the control group--the study's primary endpoint. The infarct sizes were similar across all groups. At endpoint, the mortality rates were: 63% (milrinone), 56% (enalapril) and 53% (simendan); the risk reductions were 25% (P = 0.04 vs. control) and 28% (P = 0.02 vs. control) with enalapril and simendan, respectively. Milrinone had no statistically significant effect on the survival rate. These findings suggest that, like enalapril, simendan improved survival in rats with healed myocardial infarction.

Levosimendan increases diastolic coronary flow in isolated guinea-pig heart by opening ATP-sensitive potassium channels.

Levosimendan, a novel calcium sensitizer developed for the treatment of acute heart failure, is an inodilator that increases coronary flow. Because it was recently shown that levosimendan stimulates potassium current through K(ATP) channels in isolated rat arterial cells, our aim was to assess whether the levosimendan-induced increase in coronary flow is due to the opening of the K(ATP) channels in coronary smooth muscle. The effect of levosimendan on the diastolic coronary flow velocity (DCFV) was measured in the Langendorff perfused spontaneously beating guinea-pig heart in the absence and presence of glibenclamide. Pinacidil was used as a reference compound, and the protein kinase C inhibitor bisindolylmaleimide was used to study the dilatory effect of levosimendan when the K(ATP) channels in smooth muscle are not inhibited by PKC-dependent phosphorylation. Levosimendan (0.01-1 microM) increased DCFV concentration-dependently and was noncompetitively antagonized by 0.1 microM glibenclamide, whereas pinacidil was inhibited competitively by glibenclamide. In the presence of glibenclamide the positive inotropic and chronotropic effects of levosimendan were unaltered. The effect of bisindolylmaleimide and levosimendan on DCFV was additive. The results indicate that levosimendan induced coronary vasodilation through the opening of the K(ATP) channels. Levosimendan and pinacidil probably have different binding sites on the K(ATP) channels. The additive effect of bisindolylmaleimide and levosimendan on the increase of DCFV suggests that the latter binds to the unphosphorylated form of the channel.

The role of cAMP- and cGMP-dependent protein kinases in the cardiac actions of the new calcium sensitizer, levosimendan.

OBJECTIVE: The role of phosphodiesterase III inhibition and calcium sensitization in the cardiac actions of levosimendan, (R)-[[4-(1,4,5, 6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propane dinitrile, was studied. METHODS: Various heart preparations were used to investigate positive inotropy, chromotropy, coronary flow and calcium sensitivity of contractile proteins. The cAMP- and cGMP-dependent protein kinases (PKA and PKG) were inhibited by KT5720 and KT5823, respectively. Furthermore, the synthesis of cAMP was stimulated by forskolin and increased phosphorylation of troponin I was induced by isoprenaline. RESULTS: In Langendorff guinea-pig heart, levosimendan (0.01-1 microM) and milrinone (0.1-10 microM) increased the left ventricular systolic peak pressure almost to the same extent. In the presence of KT5720 (1 microM) milrinone was devoid of positive inotropic activity. In contrast, KT5720 did not antagonize the inotropic effect of levosimendan at < or = 0.03 microM (-up to the EC50 of levosimendan). The effects of levosimendan and milrinone on heart rate and coronary flow were not affected by KT5720. The PKG inhibitor, KT5823 (1 microM), on the other hand, potentiated the levosimendan-induced increase in coronary flow while it had no effect on the increase induced by milrinone. The mechanical parameters were not affected by KT5823. In the papillary muscle, the positive inotropic effect of milrinone but not that of levosimendan was potentiated by forskolin (0.1 microM). In contrast to milrinone, the positive inotropy by levosimendan was decreased by isoprenaline pretreatment (0.1 microM; 3 min). In line with this, the calcium-sensitizing effect of levosimendan was decreased in skinned fibers prepared from isoprenaline-treated hearts. CONCLUSIONS: Our results indicate that the cardiac effects of levosimendan at its therapeutically relevant concentrations were not mediated through PKA or PKG and its positive inotropy is therefore most probably due to the previously reported troponin-C-mediated calcium sensitization of contractile proteins.

Entacapone, a novel catechol-O-methyltransferase inhibitor for Parkinson's disease, does not impair mitochondrial energy production.

Entacapone, a novel mainly peripherally acting catechol-O-methyltransferase inhibitor used in the treatment of Parkinson's disease, was evaluated for its possible uncoupling activity in cell culture, in rat liver mitochondria, and in isolated guinea-pig heart. Entacapone did not stimulate respiration in the L1210 murine T cell lymphoma cell line at the concentrations studied (5-40 microM). Furthermore, entacapone neither increased mitochondrial respiration nor impaired cardiac function at pharmacologically relevant concentrations (< 10 microM). In fact, the threshold concentration for increased mitochondrial oxygen consumption was 20 microM and half-maximal stimulation of respiration was not detected until 58 microM. Surprisingly, tolcapone, another catechol-O-methyltransferase inhibitor, which acts both peripherally and centrally, stimulated respiration in L1210 cells at the lowest concentration studied (5 microM). In addition, 1 microM tolcapone increased mitochondrial respiration, indicating that it caused uncoupling at a much lower concentration than that of 2,4-dinitrophenol, a well-known uncoupler of oxidative phosphorylation. Tolcapone also impaired the mechanical function and oxygen consumption of the isolated guinea-pig heart at 1 microM. These results show that peripherally acting entacapone, unlike the brain-penetrating tolcapone, is a safe catechol-O-methyltransferase inhibitor for the treatment of Parkinson's disease, since it does not interfere with mitochondrial energy metabolism at pharmacologically effective concentrations.

Brain regional and adrenal monoamine concentrations and behavioral responses to stress in alcohol-preferring AA and alcohol-avoiding ANA rats.

The concentrations of monoamines, precursors and metabolites in various brain regions and the levels of catecholamines in the adrenal glands were determined from naive rats of the AA and ANA lines, and from ones immediately after an escapable shock test. The brain determinations were made with a new step-gradient ion-pair elution method on a reversed phase column and coulometric detection. Several significant differences were observed in the amine concentrations, largely confirming and extending the findings made before the genetic revitalization of the lines: in particular, the AAs, unlike other alcohol-preferring rodents, had higher 5-hydroxytryptamine concentrations. The AA rats tended to have smaller changes than the ANAs in brain aminergic systems and had significantly less change in adrenal epinephrine and dopamine levels after the shock test. The AAs were consistently found to be less active than ANAs in this shock test and in a warm-water swim test, but whether this was a cause or an effect of their brain and adrenal changes could not be determined. Our behavioral results might suggest a reduced reaction of the alcohol-preferring rats to aversive stimulation.

Hippocampal rhythmic slow activity in rat lines selected for differences in ethanol-induced motor impairment.

Hippocampal rhythmic slow activity (RSA) was recorded during rotation and vibration stimulation after saline and ethanol (2 g/kg) administration in restrained alcohol-sensitive (ANT) and alcohol-insensitive (AT) rats implanted with chronic bipolar electrodes in the dorsal hippocampus. The saline-treated ANT rats had more high-frequency RSA than the AT rats, especially during the rotational stimulation of the optovestibular mechanisms. The difference was not found during ethanol sessions. Plasma corticosterone levels were significantly higher in the AT than the ANT rats after the recording sessions. This first electrophysiological demonstration of an alcohol-sensitivity difference in the brain between these rat lines is discussed in relation to behavioral tilting plane test used in the development of the lines, to the different innate responses of the lines to acute stress, and to the plausible line differences in brain GABAergic and serotonergic mechanisms that are known to modulate hippocampal EEG in rodents.

Ethanol-induced modulation of hippocampal EEG and cerebral 2-deoxyglucose accumulation in rat lines with differing alcohol sensitivities.

Rat lines (AT, alcohol tolerant, insensitive; ANT, alcohol nontolerant, sensitive) selectively bred for differences in ethanol-induced motor impairment were subjected to studies on neuronal signal processing and cerebral regional glucose uptake as influenced by an acute moderate dose (2 g/kg) of ethanol. The cerebellar membrane GABA receptor complex was also studied with binding experiments using naive AT and ANT rats. Recordings of hippocampal EEG indicated that with and without ethanol administration a rotational stimulus caused EEG synchronization in both rat lines. There was no major difference in the 2-deoxyglucose accumulation into gross brain regions between the lines. Thus, a moderate dose of ethanol does not make these rat lines distinguishable in these tests. The number of muscimol binding sites tended to be higher and that of flunitrazepam binding sites lower in the cerebellum of AT rats. These variations could not be abolished by treating the membranes with a detergent to remove possible endogenous substances bound to the receptors. The functional significance of this small difference is not known, as the relationship between receptor numbers and function is still vague. The present results indicate that the central nervous system factors involved in acute alcohol sensitivity are subtle requiring detailed study at all levels from the membrane molecules to behaving animals.