Tissue kallikrein deficiency aggravates cardiac remodelling and decreases survival after myocardial infarction in mice.

BACKGROUND: Tissue kallikrein (TK) is a major kinin-releasing enzyme present in arteries. TK is involved in cardioprotection in the setting of acute myocardial ischaemia but its role in post-ischaemic heart failure (HF), a major cause of delayed mortality after myocardial infarction (MI), is unknown. AIM: To determine whether TK deficiency in the mouse influences survival and cardiac remodelling after MI. METHODS: MI was induced in 10 week-old male TK-deficient mice and wild-type littermates. Survival was assessed up to 14 months. Cardiac morphological and functional parameters were serially measured by echocardiography. In another experiment, myocardial capillary density and NOS content were evaluated at 3 months. RESULTS: Infarct size was similar in both genotypes. MI resulted in severe cardiac dysfunction. Up to 12 months after MI, TK(-/-) mice displayed an increased mortality rate (P<0.05, relative risk of death=3.41) and aggravation of left ventricular hypertrophy and dilatation by comparison with TK(+/+) (+18% and +27% respectively, both P<0.05). NOS1 and NOS3 were abnormally regulated in the heart of TK(-/-) mice after MI. CONCLUSIONS: TK exerts a protective role in HF in mice. Coronary effects are probably involved. As partial genetic deficiency in TK activity occurs in humans, TK-deficient subjects may be at increased risk of mortality in HF.

Is it important to correct apparent drug tissue concentrations for blood contamination in the dog?

The goal of this study was to quantify in the dog the error that is made in assessing drug tissue concentrations when no correction for blood contamination is performed and hence to determine in which organs such a correction should be made. The organs investigated were the heart, the brain, the liver and the skeletal muscle, and the test drug used was the H1-antihistamine, cetirizine (0.1 or 0.6 mg/kg/day for 3 days, orally, n = 6 dogs). Radiolabelled serum albumin was used to quantitate blood trapped in the tissues. Blood and tissue samplings were performed 2 h after the last drug administration. Mean (+/-SEM) volumes of blood trapped in the liver, heart, muscle and brain were 263 +/- 12, 91 +/- 3, 27 +/- 1 and 20 +/- 2 microL/g, respectively. Apparent tissue/blood concentration ratios of cetirizine were 2.39 +/- 0.33, 1.11 +/- 0.09, 0.77 +/- 0.07 and 0.37 +/- 0.05 in the four organs. When correction for residual blood is not performed, cetirizine concentrations are underestimated (-13.6 +/- 3.2%) in the liver, slightly overestimated (+4.7 +/- 1.5 to +6.3 +/- 2.8%) in the brain, and neither over nor underestimated in the heart and muscle. Simulation data over a wide range of theoretical drug tissue/blood concentration ratios indicate that in the dog: (a) for the liver, correction of apparent tissue concentration for residual blood should be performed when the drug tissue/blood concentration ratio achieved is <0.8 or >4, (b) for the heart, correction should be made when this ratio is < or =0.4 and (c) for the brain and muscle, no correction is necessary unless the ratio is < or =0.1.

Heart rate reduction during exercise-induced myocardial ischaemia and stunning.

AIMS: The respective contributions of reduction in heart rate and inotropism in the beneficial effects of beta-blockade in ischaemic heart disease remains debated. The effects of selective heart rate reduction by ivabradine (If inhibitor) were compared to those of atenolol on exercise-induced ischaemia and stunning. METHODS AND RESULTS: In seven instrumented dogs, coronary stenosis was calibrated to suppress increase in coronary blood flow during a 10-min treadmill exercise. When administered before exercise, atenolol and ivabradine similarly reduced heart rate versus saline at rest and during exercise (154+/-2 and 155+/-9 vs 217+/-13 beats/min, respectively). During exercise, left ventricular wall thickening (LVWth) was reduced to 2+/-1% from 23+/-4% under saline but ivabradine limited this effect (10+/-3%) and reduced the subsequent myocardial stunning vs saline. Atenolol also limited LVWth decrease during exercise (17+/-4%) but had no effect during recovery. When administered after exercise, ivabradine attenuated stunning and this effect disappeared when heart rate reduction was corrected by atrial pacing. Atenolol administered after exercise severely depressed LVWth vs saline. CONCLUSION: Selective heart rate reduction not only provides an anti-ischaemic effect but also per se improves contractility of the stunned myocardium. Additional negative inotropism is protective against ischaemia but deleterious during stunning.

Haemodynamic effects of dual blockade of the renin-angiotensin system in spontaneously hypertensive rats: influence of salt.

OBJECTIVE: To elucidate the mechanisms responsible for the adverse renal effects induced by dual blockade of the renin-angiotensin system (RAS) and the role of salt therein. METHODS: The effects of enalapril, losartan and their combination on blood pressure, renal haemodynamics, renal function and RAS were investigated over a wide range of doses in spontaneously hypertensive rats fed either a low-sodium or a high-sodium diet. RESULTS: In rats fed the low-sodium diet, the losartan-enalapril combination induced the same dose-dependent haemodynamic and hormonal changes as did three- to 10-fold greater doses of enalapril or losartan alone. When a strong decrease (> 50%) in blood pressure was achieved (with 10 mg/kg enalapril plus 10 mg/kg losartan, 100 mg/kg enalapril or 100 mg/kg losartan), a massive renal vasoplegia occurred and renal insufficiency developed. In addition, because of the huge release of renin, angiotensinogen concentrations were reduced, leading to a decrease in intrarenal angiotensins. In rats fed the high-sodium diet, those treated with the enalapril 30 mg/kg plus losartan 30 mg/kg combination, despite complete functional RAS blockade, exhibited smaller decreases in blood pressure and renal resistance, lesser release of renin and angiotensinogen consumption, and a normal renal function. These effects were similar to those produced by 100 mg/kg of enalapril or losartan in rats fed the high-salt diet, or by 10 mg/kg of enalapril or of losartan in rats fed the low-salt diet. CONCLUSIONS: Dual RAS blockade could be either beneficial, when sodium intake is unrestricted, or dangerous, when sodium intake is restricted.

Effects of angiotensin II type 1 receptor blockade in ApoE-deficient mice with post-ischemic heart failure.

We investigated in mice whether atherosclerosis exacerbates the development of post-ischemic heart failure and alters the beneficial effects of long-term angiotensin II type 1 receptor blockade in this model. ApoE-deficient (ApoE(-/-)) and C57BL/6J (C57) mice with myocardial infarction (coronary ligation) received vehicle (C57 and ApoE(-/-)) or irbesartan (Ir, 50mg/kg/d orally, C57-Ir and ApoE(-/-)-Ir). Ten months post myocardial infarction, survival rates were similar in C57 (58%) and ApoE(62%). Atherosclerosis induced no significant alteration in blood pressure, cardiac output (fluospheres), total peripheral resistance, or shortening fraction (echocardiography) but increased renal resistance (+50%, P<0.05). Chronic Ir treatment significantly improved survival to a similar extent in both C57-Ir (85%) and ApoE(-/-)-Ir (86%). It also decreased blood pressure to a similar extent in both strains (-16% and -18%, both P<0.05). In C57-Ir mice, Ir did not modify cardiac output or total peripheral resistance, but it decreased renal resistance (-28%, P<0.001) and left-ventricular weight (-28%, P<0.05). In ApoE(-/-)-Ir mice, Ir limited atherosclerotic lesions (-13%, P<0.05), increased cardiac output (+28%, P<0.05) and shortening fraction (+24%, P<0.05), and decreased total peripheral resistance (-33%, P<0.01), renal resistance (-61%, P<0.001), and left-ventricular weight (-27%, P<0.001). In conclusion, atherosclerosis does not worsen heart failure development in mice and, although the beneficial cardiovascular effects of AT1 receptor blockade are greater in ApoE(-/-) than in C57, reduction in mortality is similar in both strains.

Comparative pharmacokinetics and pharmacodynamics of intravenous and oral nefopam in healthy volunteers.

To determine the pharmacokinetic, subjective effects of a single 20 mg dose of nefopam administered either intravenously or orally in healthy volunteers, twenty-four healthy Caucasian men received 20 mg nefopam orally+placebo intravenous infusion and placebo orally+intravenous infusion of 20 mg nefopam with one week interval, in a double-blind, double-dummy cross-over study. Nefopam and desmethyl-nefopam plasma concentrations were measured by HPLC with UV detection up to 48 hr after drug administration. Self-rating questionnaires (Mood and vigilance Visual Analogue Scales, Addiction Research Centre Inventory) and drug safety were investigated. The F value (bioavailability) of the parent drug was 0.36+/-0.13. The AUCoral/AUCiv ratio of nefopam+desmethyl-nefopam was 0.62+/-0.23. The half-life of nefopam was similar whether administered orally (5.1+/-1.3 hr) or intravenously (5.1+/-0.6 hr). The half-life of desmethyl-nefopam was two to three times longer than that of the parent molecule (orally: 10.6+/-3.0 versus 5.1+/-1.3 hr, P<10(-4) and intravenously: 15.0+/-2.4 versus 5.1+/-0.6 hr, P<10(-4)). As assessed by the Addiction Research Centre Inventory, no evidence of abuse liability in healthy, drug-naive volunteers was observed. On visual analogue scales, volunteers rated themselves as more drowsy, less alert, less energetic and less anxious after oral compared to intravenous administration. The AUC0-->24 hr of anxiety and energy parameters were not different after oral and intravenous administration: 90+/-142 versus 35+/-84 (P=0.27) and 66+/-74 versus 46+/-54 mm x hr (P=0.36), respectively. The AUC0-->24 hr of drowsiness and alertness parameters were significantly greater after oral than after intravenous administration: 68+/-65 versus 27+/-30 (P=0.005) and 54+/-63 versus 28+/-48 mm x hr (P=0.03), respectively. A clockwise hysteresis loop was observed for drowsiness in 16 out of 24 volunteers after oral administration. The results suggest that in healthy volunteers desmethyl-nefopam may contribute to the pharmacodynamic effects of single dose nefopam solution administered orally. This study shows a rather low bioavailability of nefopam given in intravenous solution when administered orally. Nevertheless, when the main metabolite desmethyl-nefopam is taken into account, the ratio of the areas under the curves is almost doubled.

Fluvastatin prevents renal dysfunction and vascular NO deficit in apolipoprotein E-deficient mice.

OBJECTIVE: The objective of this study was to investigate the effects of fluvastatin on atherosclerosis, systemic and regional hemodynamics, and vascular reactivity in apolipoprotein E-deficient (ApoE(-/-)) mice. METHODS AND RESULTS: Hemodynamics (fluospheres) and vasomotor responses of thoracic aorta and carotid artery were evaluated in male wild-type (WT) and untreated (ApoE(-/-) Control) or fluvastatin-treated (50 mg/kg per day for 20 weeks) ApoE(-/-) mice, all fed a Western-type diet. Plasma cholesterol and aortic root atherosclerotic lesions (ALs) were greater in ApoE(-/-) Control mice (19+/-1 mmol/L and 63,0176+/-38,785 micro m(2), respectively) than in WT mice (2+/-1 mmol/L and 1+/-1 micro m(2), respectively, P<0.01). Fluvastatin significantly decreased plasma cholesterol (-53%) but failed to limit ALs. Renal blood flow was significantly reduced in ApoE(-/-) Control versus WT (-25%, P<0.05) mice. This reduction was prevented by fluvastatin. Aortic and carotid endothelium-dependent relaxations to acetylcholine were not altered in ApoE(-/-) Control versus WT mice. In carotid arteries from WT mice, these responses were abolished after nitro-L-arginine (L-NA), whereas those from ApoE(-/-) Control were only partially inhibited after L-NA but fully abolished after L-NA+diclofenac. Thus, in carotid arteries from ApoE(-/-) mice, vasodilating prostanoids compensate the deficit in NO availability. Fluvastatin prevented this carotid NO deficit. CONCLUSIONS: In ApoE(-/-) mice, chronic fluvastatin treatment preserved renal perfusion and vascular NO availability independently from atherosclerotic lesion prevention.

Contributions of heart rate and contractility to myocardial oxygen balance during exercise.

The respective contributions of heart rate (HR) reduction and left ventricular (LV) negative inotropy to the effects of antianginal drugs are debated. Accordingly, eight instrumented dogs were investigated during exercise at spontaneous and paced HR (250 beats/min) after administration of either saline, atenolol, or ivabradine (selective pacemaker current channel blocker). During exercise, atenolol and ivabradine (both 1 mg/kg iv) similarly reduced HR (-30% from 222 +/- 5 beats/min), and LV mean ejection wall stress was not altered. LV dP/dt(max) was reduced by atenolol but not ivabradine. Diastolic time (DT) was increased by atenolol versus saline (195 +/- 6 vs. 123 +/- 4 ms, respectively) and to a greater extent by ivabradine (233 +/- 11 ms). Myocardial oxygen consumption (MVo(2)) was lower under ivabradine and atenolol versus saline (6.7 +/- 0.6 and 4.7 +/- 0.4 vs. 8.1 +/- 0.6 ml/min, respectively, P < 0.05). Under pacing, DT and MVo(2) were similar between ivabradine and saline but significantly reduced with atenolol. Thus HR reduction and negative inotropy equally contribute to the reduction in MVo(2) during exercise in the normal heart. The negative inotropy limits the increase in DT afforded by HR reduction.

Reperfusion duration paradox with late myocardial preconditioning in rabbits.

In this study, pharmacological late preconditioning was induced in 53 rabbits with an adenosine A(1) receptor agonist (2-chloro-N(6)-cyclopentyladenosine, CCPA, 100 microg/kg), or a NO-donor (S-nitroso-N-acetyl-penicillamine, SNAP, 2.5 microg/kg/min; 75 min) vs. saline as control. Later, after 24 h, rabbits underwent a 30-min coronary occlusion and subsequent reperfusion. After 3 h of coronary artery reperfusion, infarct size was reduced with CCPA (43+/-4%) and SNAP (27+/-4%) vs. saline (56+/-4%). However, after 72 h of coronary artery reperfusion, infarct sizes were similar in all groups, demonstrating an only transient effect of late preconditioning against myocardial infarction. Combined administration of CCPA and SNAP failed to induce sustained cardioprotection.

Role of microvascular rarefaction in the increased arterial pressure in mice lacking for the endothelial nitric oxide synthase gene (eNOS3pt-/- ).

OBJECTIVES: Mechanisms involved in hypertension in homozygous mice for the defective endothelial nitric oxide synthase gene (eNOS-/-) have not been fully elucidated. As NO is a potent vasodilator agent and possibly promotes angiogenesis, we investigated whether vasoconstriction and/or microvascular rarefaction could explain hypertension in these mice. METHODS: Immunohistochemistry with mouse monoclonal smooth muscle alpha-actin antibody was used to detect arterioles, and quantification of arteriolar density was performed in the left ventricle and in the gracilis muscle of 12-week-old male eNOS+/+ and eNOS-/- mice. Haemodynamic parameters - mean arterial pressure (MAP), cardiac index (CI), total peripheral résistance (TPR), myocardial blood flow, muscular blood flow and corresponding resistances - were measured or calculated using the fluorescent microsphere method in basal conditions and after infusion of sodium nitroprusside (SNP) (5 to 150 microg/kg per min) in eNOS-/- mice, compared with eNOS+/+ mice. RESULTS: We evidenced a significant decrease in arteriolar density in the heart (-16%, P < 0.02) and in the gracilis muscle (-22%, P < 0.05) in eNOS-/- mice. In basal conditions, eNOS-/- mice developed significant hypertension (MAP = 127 +/- 14 versus 77 +/- 14 mmHg, P < 0.001) associated with decreased CI (-29%, P < 0.001) and increased TPR (+ 125%, P < 0.001). Coronary and gracilis muscular resistances were increased (by 75 and 89% respectively, P < 0.001) compared with eNOS+/+ mice, whereas myocardial and skeletal muscle tissue blood flows were not affected. After SNP administration (10 microg/kg per min), a dose that did not significantly modify haemodynamic parameters in eNOS+/+ mice, MAP, TPR and regional resistances were normalized in eNOS-/- mice, showing that vasodilation may correct hypertension in eNOS-/- mice. However, under maximal vasodilating conditions, TPR and regional resistances remained significantly higher in eNOS-/- mice than those of eNOS+/+ mice. CONCLUSION: Anatomical and functional results show that both vasoconstriction and arteriolar rarefaction are involved in hypertension of eNOS-/- mice. Indeed, under maximal vasodilation, arterial pressure and TPR remained significantly higher in eNOS-/- mice than in eNOS+/+ mice, evidencing a major role of microvascular rarefaction in this model of hypertension.

Cardiovascular phenotypes of kinin B2 receptor- and tissue kallikrein-deficient mice.

To clarify the role of the kallikrein-kinin system in cardiovascular homeostasis, the systemic and regional hemodynamics of kinin B2 receptor-deficient (B2-/-) and tissue kallikrein-deficient (TK-/-) mice were compared with their wild-type (WT) littermates on a pure C57BL/6 genetic background. B2-/-, TK-/-, and WT adult mice were normotensive and displayed normal hemodynamic (left ventricular [LV] pressure, cardiac output, total peripheral resistance, dP/dt(max)) and echocardiographic (septum and LV posterior wall thickness, LV diameter, LV mass, and LV fractional shortening) parameters. However, heart rate was lower in B2-/- mice compared with TK-/- and WT mice. In addition, B2-/- mice, but not TK-/- mice, exhibited lower coronary and renal blood flows and greater corresponding vascular resistances than did WT mice, indicating a tonic physiological vasodilating effect of bradykinin in these vascular beds. However, maximal coronary vasodilatation capacity, estimated after dipyridamole infusion, was similar in the 3 groups of mice. B2-/- mice were significantly more sensitive than were TK-/- mice to the vasoconstrictor effects of angiotensin II and norepinephrine. Finally, renin mRNA levels were significantly greater in B2-/- mice and smaller in TK-/- mice compared with WT mice. Taken together, these results indicate that under basal conditions, the kinin B2 receptor is not an important determinant of blood pressure in mice but is involved in the control of regional vascular tone in the coronaries and the kidneys. The phenotypic differences observed between TK-/- and B2-/- mice could be underlain by tissue kallikrein kinin-independent effect and/or kinin B1 receptor activation.

Adenosine A(1)-receptor induced late preconditioning and myocardial infarction: reperfusion duration is critical.

We investigated the influence of coronary artery reperfusion (CAR) duration on the infarct-limiting properties of adenosine A(1)-receptor stimulation-induced delayed preconditioning (A(1)-DPC) compared with ischemia-induced delayed preconditioning (I-DPC). Sixty-one chronically instrumented conscious rabbits successfully underwent the following protocol. On day 1, rabbits were randomly divided into four groups: control (saline, iv), I-DPC (six 4-min coronary artery occlusion/4-min reperfusion cycles), A(1)-DPC(100) (N(6)-cyclopentyladenosine, 100 microg/kg iv), and A(1)-DPC(400) (N(6)-cyclopentyladenosine, 400 microg/kg iv). On day 2 (i.e., 24 h later), rabbits underwent a 30-min coronary artery occlusion after which CAR was started and maintained for either 3 or 72 h. Infarct size (percentage of the area at risk) was determined by triphenyltetrazolium chloride staining. After 3 h of CAR, I-DPC, A(1)-DPC(100), and A(1)-DPC(400) significantly decreased infarct size (36 +/- 5, 41 +/- 4, 38 +/- 5%, respectively) compared with control (55 +/- 3%). After 72 h of CAR, infarct sizes were not significantly different among the four groups. This result was confirmed by histologic analysis. Thus A(1)-DPC at the two investigated doses, as well as I-DPC, decreased infarct size after 3 h but not 72 h of CAR.

Differential effects of heart rate reduction and beta-blockade on left ventricular relaxation during exercise.

Left ventricular (LV) relaxation is crucial for LV function, especially during exercise. We compared the effects of increasing doses of ivabradine, a selective inward hyperpolarization-activated current inhibitor, and atenolol on the rate and extent of LV relaxation (best fit method: time constant tau(BF), pressure asymptote P(BF)) at rest and during exercise. Eight dogs were chronically instrumented to measure LV pressure and LV wall stresses. During exercise under saline, heart rate increased from 108 +/- 5 to 220 +/- 6 beats/min and tau(BF) was significantly reduced from 22 +/- 1 to 14 +/- 2 ms. At rest, atenolol but not ivabradine increased tau(BF). For similar heart rate reductions during exercise, atenolol impeded the shortening of tau(BF) (23 +/- 2 ms) whereas ivabradine had no effect (15 +/- 2 ms). The extent of the relaxation process (P(BF)) at peak exercise was increased by ivabradine, and to a greater extent by atenolol, compared with saline. Thus, for a similar reduction in heart rate at rest and during exercise, ivabradine, in contrast with atenolol, does not exert any negative lusitropic effect.