Jacques Mizrahi

Increased Protein Kinase C Activity and Expression of Ca2+-Sensitive Isoforms in the Failing Human Heart

BACKGROUND Increased expression of Ca2+-sensitive protein kinase C (PKC) isoforms may be important markers of heart failure. Our aim was to determine the relative expression of PKC-beta1, -beta2, and -alpha in failed and nonfailed myocardium. METHODS AND RESULTS Explanted hearts of patients in whom dilated cardiomyopathy or ischemic cardiomyopathy was diagnosed were examined for PKC isoform content by Western blot, immunohistochemistry, enzymatic activity, and in situ hybridization and compared with nonfailed left ventricle. Quantitative immunoblotting revealed significant increases of >40% in PKC-beta1 (P<0.05) and -beta2 (P<0.04) membrane expression in failed hearts compared with nonfailed; PKC-alpha expression was significantly elevated by 70% in membrane fractions (P<0.03). PKC-epsilon expression was not significantly changed. In failed left ventricle, PKC-beta1 and -beta2 immunostaining was intense throughout myocytes, compared with slight, scattered staining in nonfailed myocytes. PKC-alpha immunostaining was also more evident in cardiomyocytes from failed hearts with staining primarily localized to intercalated disks. In situ hybridization revealed increased PKC-beta1 and -beta2 mRNA expression in cardiomyocytes of failed heart tissue. PKC activity was significantly increased in membrane fractions from failed hearts compared with nonfailed (1021+/-189 versus 261+/-89 pmol. mg-1. min-1, P<0.01). LY333531, a selective PKC-beta inhibitor, significantly decreased PKC activity in membrane fractions from failed hearts by 209 pmol. min-1. mg-1 (versus 42.5 pmol. min-1. mg-1 in nonfailed, P<0.04), indicating a greater contribution of PKC-beta to total PKC activity in failed hearts. CONCLUSIONS In failed human heart, PKC-beta1 and -beta2 expression and contribution to total PKC activity are significantly increased. This may signal a role for Ca2+-sensitive PKC isoforms in cardiac mechanisms involved in heart failure.

The Nitric Oxide Donor ITF 1129 Augments Subendocardial Blood Flow During Exercise-Induced Myocardial Ischemia

The effect of the nitric oxide donor ITF 1129 and nitroglycerin (NTG) on myocardial blood flow was examined in dogs with a Doppler velocity probe, hydraulic occluder, and indwelling microcatheter in the left anterior descending coronary artery (LAD). Studies were performed during treadmill exercise in the presence of a coronary artery stenosis. The effects of ITF 1129 in doses of 3 and 10 micrograms/kg/min i.v. were compared with NTG (2 micrograms/kg/min i.v.). Neither ITF 1129 nor NTG caused significant alteration of heart rate, arterial blood pressure, or left ventricular systolic pressure. During partial inflation of the occluder to decrease distal coronary pressure to 55 +/- 2 mm Hg, mean myocardial blood flow measured with microspheres was 0.72 +/- 0.14 ml/min/g in the region perfused by the stenotic coronary artery compared with 2.93 +/- 0.40 ml/min/g in a normally perfused control region. With no change in distal coronary pressure, ITF 1129 increased mean myocardial blood flow in the stenosis perfused region to 1.15 +/- 0.24 ml/min/g (3 micrograms/kg/min i.v.) and to 1.20 +/- 0.28 ml/ min/g (10 micrograms/kg/min i.v.), whereas NTG (2 micrograms/kg/min iv) increased blood flow to 1.16 +/- 0.22 ml/min/g (each p < 0.05). The increase in myocardial blood flow produced by ITF 1129 or NTG occurred principally in the deeper myocardial layers with no change in subepicardial flow. As a result, the subendocardial/subepicardial blood flow ratio (ENDO/EPI) increased from 0.44 +/- 0.09 during control stenosis to 0.85 +/- 0.13 after ITF 1129 (10 micrograms/kg/min i.v.) and to 0.81 +/- 0.12 after NTG. Neither ITF 1129 nor NTG significantly altered myocardial blood flow in the normally perfused control region. The effect of ITF 1129 and NTG on myocardial perfusion occurred without alterations of distal coronary pressure or left ventricular diastolic pressure, indicating a primary effect on the intramural coronary microvasculature.

Nitrovasodilators ITF 296 and isosorbide dinitrate exert antiischemic activity by dilating coronary penetrating arteries.

We examined the effect of the novel nitrovasodilator ITF 296 and isosorbide dinitrate (ISDN) on myocardial blood flow (BF) distal to a coronary artery stenosis. Eleven dogs with a Doppler velocity probe, hydraulic occluder, and indwelling microcatheter in the left anterior descending coronary artery (LAD) were studied during treadmill exercise in the presence of a coronary artery stenosis. On separate days, the effects of ITF 296 in doses of 4 and 20 μg/kg/min i.v. or ISDN 20 μg/kg/min i.v. were compared. Coronary pressure distal to the stenosis was maintained constant during the control period and after administration of either nitrovasodilator. Neither ITF 296 nor ISDN significantly altered heart rate (HR), arterial blood pressure (BP), or left ventricular enddiastolic pressure (LVEDP). In the presence of a stenosis that decreased distal coronary pressure to 58 ± 4 mm Hg, mean myocardial BF measured with microspheres was 0.91 ± 0.08 ml/min/g in the LAD-dependent region and 2.36 ± 0.11 ml/min/g in the posterior control region, respectively. With no change in distal coronary pressure, ITF 296 increased mean BF in the LAD region to 1.25 ± 0.05 ml/min/g (4 μg/kg/min i.v.) and 1.40 ± 0.10 ml/min/g (20 μg/kg/min i.v.), whereas ISDN (20 μg/kg/min i.v.) increased flow to 1.28 ± 0.18 ml/min/g (each p < 0.05). The increase in BF occurred exclusively in the deeper layers, with no change in subepicardial BF. Consequently, the endocardial/epicardial (endo/epi) BF ratio increased from 0.33 ± 0.04 during control stenosis to 0.70 ± 0.10 after ITF 296 (20 μg/kg/min), and to 0.56 ± 0.08 after ISDN (each p < 0.05). Neither ITF 296 nor ISDN had an effect on myocardial BF in the normally perfused control region. Therefore, both ITF 296 and ISDN improved BF to the deeper myocardial layers distal to a coronary artery stenosis. This effect occurred without alterations in stenosis severity or diastolic intraventricular pressure, suggesting that these agents act by dilating the penetrating arteries which deliver BF to the subendocardium.

Protective effects of ITF 296 in the isolated rabbit heart subjected to global ischemia.

The anti-ischemic action of ITF 296 was evaluated in isovolumic, electrically driven rabbit heart preparations subjected to temporary ischemia and reperfusion. Reduction of perfusion rate from 20 to 0.2 ml/min produced a sharp decrease of peak systolic pressure, left ventricular (LV)-developed pressure, and LV dP/dt, which culminated in complete ventricular arrest within 3–4 min. Thereafter, LV end-diastolic pressure (LVEDP) progressively increased, suggesting a severe ischemic episode. Reperfusion with 20 ml Krebs solution/min after 40 min of low-flow perfusion produced only minimal recovery from the rhythm disturbances associated with cardiac mechanical activity. During reperfusion, loss of myocardial elasticity was associated with a significant increase in coronary vascular resistance, as indicated by the augmentation of coronary perfusion pressure. Injection of ITF 296 (0.3–10 μM) into the perfusion system dose-dependently inhibited the increase in LVEDP that took place during ischemia and progressively improved the recovery of a regular rhythm in the isolated heart. Isosorbide dinitrate (ISDN) at a concentration of 10 μM exerted a protective action on the myocardium similar to that obtained with ITF 296 (3 μM). Treatment with ITF 296 (1 and 10 μM) resulted in a dose-dependent increase in the rate of 6-keto-PGF 1α biosynthesis during both the ischemia and the reperfusion period (+157% over basal values). Similar results were obtained when hearts were pretreated with ISDN (10 μM). Infusion of a buffer containing NG-monomethyl-L-arginine (L-NMMA: NO synthase inhibitor at 10 μM) just before reduction of flow, markedly exacerbated the effects of ischemia and reperfusion on the myocardium and increased coronary perfusion pressure. The effects of L-NMMA were clearly antagonized by ITF 296 (10 μM) or L-arginine (100 μM). Mechanical activity and sinus rhythm during reperfusion were rapidly and completely restored, and coronary resistance values were close to the preischemic values. As with ISDN, ITF 296 significantly increased the rate of synthesis of 6-keto-PGF1α both under basal conditions and during reperfusion.

Safety and pharmacologic activity of a new nitrate ester, ITF 296, after intravenous administration in healthy volunteers.

To evaluate the safety and pharmacologic activity of ITF 296 in humans, three groups of healthy male normotensive subjects were studied. The first two groups (six subjects each) received, in ascending order, three dose levels of ITF 296 by 30-min intravenous infusion (group I, 0.1, 0.5, 1.0 μg/kg/min; group II, 2.0, 4.0, 6.0 μg/kg/min). The third group of eight subjects received, in ascending order, four dose levels of ITF 296 (10, 20, 40, 80 μg/kg) by 1-min i.v. injection. The study was double-blind, and placebo-controlled according to a within-patient, incomplete, unbalanced block design, such that each subject received the placebo once. Hemodynamics were assessed by means of Dynamap and BOMED. The following parameters were evaluated at different times before and after ITF 296 administration: systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), stroke volume index (SVI), cardiac index (CI), and systemic vascular resistance index (SVRI). Blood samples for kinetic assessment of ITF 296 were taken before and at different times after ITF 296 administration. The drug was well tolerated. Only a few mild (except for one, moderate) side effects (mainly headache and dizziness) were reported, usually at the higher dose levels. All safety clinical chemistry and hematologic parameters were unaffected. After i.v. infusion of ITF 296, blood pressure started to fall at the dose of 2 μg/kg/min, DBP being significantly reduced at doses above 1 μg/kg/min. The effect lasted for up to 60 min after the end of the infusion. The increase in heart rate was only modest, although apparently dose-dependent. SVI was only slightly reduced, and the other hemodynamic parameters did not change. After bolus administration of ITF 296. SBP was significantly reduced starting at a dose of 20 μg/kg with higher doses producing a more marked effect (up to – 15 mm Hg), DBP was significantly reduced only at the higher dose level of 80 μg/kg. The effect lasted for up to 60 min after bolus administration. HR was slightly increased after doses of 40 and 80 μg/kg. SVI was slightly reduced and a small transient decrease in CI was observed, whereas SVRI did not change. Satisfactory, linear kinetic correlation was found between total doses administered and AUCs measured. ITF 296 in healthy male normotensive volunteers was effective and well tolerated. The results of this study justify the planning of further studies in patients in order to test the anti-ischemic activity of the compound.

Mechanism of ITF 296-induced vasorelaxation compared to nitroglycerin and isosorbide dinitrate: relationship between relaxation of rabbit aorta and tissue cGMP.

The vasorelaxant properties of ITF 296, a new mononitrate ester, were studied in endothelium-denuded rabbit aortic rings and were compared to nitroglycerin (NTG) and isosorbide dinitrate (ISDN). In norepinephrine-contracted arteries, ITF 296, NTG, and ISDN elicited maximal and concentration-dependent vasodilatation with pD2 values of 7.07, 7.95, and 7.2, respectively. The concentration-relaxation curves of ITF 296 were shifted markedly to the right (p < 0.01) in the presence of 10 μM methylene blue (MB) and 3 μM oxyhemoglobin (HbO2), whereas a significant shift to the left (p < 0.01) was observed in the presence of 10 μM M&B-22948 (a specific cGMP phosphodiesterase inhibitor). When KCI (60 mM) was used as contracting agent, a weak relaxation was observed with ITF 296, suggesting the absence of activity on the voltage-dependent Ca2+ channels. A time-dependent increase in cGMP content and a positive correlation between cGMP and vasodilatation were observed in norepinephrine-contracted arteries after exposure to a single submaximal concentration of ITF 296 (1 μM). Similar results were obtained with NTG and ISDN, although NTG was found to be more active than ITF 296 or ISDN. The presence of either MB or HbO2 almost completely abolished the increase in cGMP induced by ITF 296, whereas a further increase in cGMP was observed in the presence of isobutylmethylxanthine. No changes in cAMP levels were observed after exposure of the tissues to a concentration of ITF 296 that induced significant elevation in the cGMP content. In the presence of L-cysteine, ITF 296 stimulated semipurified rat lung guanylate cyclase at higher concentrations than those of NTG or ISDN, probably because of its lower rate of nitric oxide (NO) release. These results suggest that, in common with the reference compounds NTG and ISDN, ITF 296-induced vasorelaxation in rabbit aortic rings is mediated by an NO-cGMP mechanism.

The discovery of a new organic nitrate: an overview.

Organic nitrates have been in therapeutic use for the treatment of angina pectoris for over a century. During the past decade, the search for new organic nitro esters with reduced side effects and improved oral bioavailability has been greatly intensified. We have discovered a new class of organic nitrates characterized by good oral activity and a coronary vascular selectivity greater than that of glyceryl trinitrate. Full structure-activity relationship studies of this new class of nitroesters are reported. From the screening of these compounds, ITF 296 was chosen for further evaluation.

Protective Effect of the Nitrate Ester ITF 296 Against: Methacholine-Induced Myocardial Ischemia in the Anesthetized Rat

The activity of ITF 296 against methacholine-induced myocardial ischemia was investigated in anesthetized rats in comparison with the organic nitrates nitroglycerin (NTG) and isosorbide dinitrate (ISDN), the K+ -channel openers nicorandil and cromakalim, the Ca2+ -channel blocker amlodipine, and the vasodilator dipyridamole. Given as i.v. boluses, ITF 296 (0.1–100 μg/kg) dose-dependently prevented methacholine-induced ST-segment elevation without affecting mean arterial blood pressure or heart rate to a significant extent. In this situation, ITF 296 was 10− to 30-fold more potent than the other coronary vasodilators tested. The protective effect of ITF 296 and ISDN against myocardial ischemia was also observed after oral administration, demonstrating good absorption and a limited first-pass metabolism of the two drugs. The anti-ischemic action of ITF 296 at 1 and 10 μg/kg/min was well maintained during 2 h of continuous i.v. infusion, whereas the effect of NTG and ISDN was attenuated or abolished by the end of the infusion. The new nitrate ester ITF 296 has a potent and long-lasting cardioprotective action in the anesthetized rat. The anti-ischemic effect displayed by this compound is probably mediated by an improvement of myocardial blood supply caused by pronounced coronary dilatation, whereas the contribution of systemic vasodilation is not important. Moreover, the maintenance of the anti-ischemic action during continuous i.v. infusion suggests a reduced “tolerance” development for ITF 296 compared with other nitrovasodilators.

Pharmacokinetics of Isosorbide Dinitrate in Healthy Volunteers After 24-Hour Intravenous Infusion

No studies have examined the pharmacokinetics of isosorbide dinitrate (ISDN) after infusion of long duration, even though such infusions are used in patients. We therefore measured ISDN and its active metabolites, isosorbide‐5‐mononitrate (IS5MN) and isosorbide‐2‐mononitrate (IS2MN), in plasma of 9 healthy volunteers who received a continuous intravenous infusion of ISDN for 24 hours at a dose rate that lowered diastolic blood pressure by 10% during the first 30 minutes of infusion. All subjects tolerated the infusion except one who experienced intolerable headache. Five subjects received 1 μg · min−1 · kg−1, one 2 μg · min−1 · kg−1, and two 4 μg · min−1 · kg−1 ISDN, whereas the full rate of 6 μg · min−1 · kg−1 was used continuously in one subject. At all infusion rates the plasma concentrations of ISDN were higher at 24 hours than at earlier times, suggesting that a steady‐state condition had not been reached at that time. The same was true for the mononitrate metabolites, which reached higher plasma concentrations and were cleared more slowly than the parent compound after the end of the infusion. Apparent elimination half‐lives of ISDN, IS2MN, and IS5MN were 67 ± 10 minutes, 115 ± 13 minutes, and 272 ± 38 minutes, respectively. Comparison of low‐rate infusions (1 and 2 μg · min−1 · kg−1) with high‐rate infusions (4 and 6 μg · min−1 · kg−1) showed that the plasma concentration ratios at 24 hours of mononitrate metabolites to parent drug and apparent plasma clearance of ISDN were almost halved at the higher infusion rates.

Coronary Haemodynamic Profile of the New Nitro-Ester Derivative ITF 296 in the Conscious Dog

The action of low doses of nitrovasodilators on coronary circulation is characterised by a selective dilatation of large conductance coronary arteries1,2,3, which is accompanied by a minimal effect on small coronary resistance vessels4,5,6,7. This ability of nitroderivatives to dilate selectively large coronary arteries, becomes of major importance when the capability of the conduit epicardial coronary arteries to dilate in response to increases in blood flow is impaired, as it happens after removal of coronary endothelium with angioplasty8 or in patients with atherosclerosis9,10. In those conditions, while the flow-dependent endothelium-mediated response is depressed, the endothelium-independent relaxing effect of nitravasodilators on large conduit coronary arteries remains effective. This has been confirmed in clinical studies, where the coronary vasospasm in anginal patients in which endothelium is likely not functional, has been effectively reversed by nitrovasodilators 11,12