Role of kinins in chronic heart failure and in the therapeutic effect of ACE inhibitors in kininogen-deficient rats

Using Brown Norway Katholiek (BNK) rats, which are deficient in kininogen (kinin precursor) due to a mutation in the kininogen gene, we examined the role of endogenous kinins in 1) normal cardiac function; 2) myocardial infarction (MI) caused by coronary artery ligation; 3) cardiac remodeling in the development of heart failure (HF) after MI; and 4) the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEI) on HF after MI. Two months after MI, rats were randomly treated with vehicle or the ACEI ramipril for 2 mo. Brown Norway rats (BN), which have normal kininogen, were used as controls. Left ventricular (LV) end-diastolic volume (EDV), end-systolic volume (ESV), end-diastolic pressure (EDP), and ejection fraction (EF) as well as myocardial infarct size (IS), interstitial collagen fraction (ICF), cardiomyocyte cross-sectional area (MCA), and oxygen diffusion distance (ODD) were measured. We found that 1) cardiac hemodynamics, function, and histology were the same in sham-ligated BN and BNK rats; 2) IS was similar in BN and BNK; 3) in rats with HF treated with vehicle, the decrease in LVEF and the increase in LVEDV, LVESV, LVEDP, ICF, MCA, and ODD did not differ between BNK and BN; and 4) ACEI increased EF, decreased LVEDV and LVESV, and improved cardiac remodeling in BN-HF rats, and these effects were partially blocked by the bradykinin B(2) receptor antagonist icatibant (HOE-140). In BNK-HF rats, ACEI failed to produce these beneficial cardiac effects. We concluded that in rats, lack of kinins does not influence regulation of normal cardiac function, myocardial infarct size, or development of HF; however, kinins appear to play an important role in the cardioprotective effect of ACEI, since 1) this effect was significantly diminished in kininogen-deficient rats and 2) it was blocked by a B(2) kinin receptor antagonist in BN rats.     

Cardiac kinin level in experimental diabetes mellitus: role of kininases

Diabetes mellitus impairs the cardiac kallikrein-kinin system by reducing cardiac kallikrein (KLK) and kininogen levels, a mechanism that may contribute to the deleterious outcome of cardiac ischemia in this disease. We studied left ventricular (LV) function and bradykinin (BK) coronary outflow in buffer-perfused, isolated working hearts (n = 7) of controls and streptozotocin (STZ)-induced diabetic rats before and after global ischemia. With the use of selective kininase inhibitors, the activities of angiotensin I-converting enzyme, aminopeptidase P, and neutral endopeptidase were determined by analyzing the degradation kinetics of exogenously administered BK during sequential coronary passages. Basal LV function and coronary flow were impaired in STZ-induced diabetic rats. Neither basal nor postischemic coronary BK outflow differed between control and diabetic hearts. Reperfusion after 15 min of ischemia induced a peak in coronary BK outflow that was of the same extent and duration in both groups. In diabetic hearts, total cardiac kininase activity was reduced by 41.4% with an unchanged relative kininase contribution compared with controls. In conclusion, despite reduced cardiac KLK synthesis, STZ-induced diabetic hearts are able to maintain kinin liberation under basal and ischemic conditions because of a primary impairment or a secondary downregulation of kinin-degrading enzymes.     

Regulation of components of the brain and cardiac renin-angiotensin systems by 17beta-estradiol after myocardial infarction in female rats

The present study tested the hypothesis that 17beta-estradiol (E2) inhibits increases in angiotensin-converting enzyme (ACE) and ANG II type 1 receptor (AT1R) in the brain and heart after myocardial infarction (MI) and, thereby, inhibits development of left ventricular (LV) dysfunction after MI. Age-matched female Wistar rats were treated as follows: 1) no surgery (ovary intact), 2) ovariectomy + subcutaneous vehicle treatment (OVX + Veh), or 3) OVX + subcutaneous administration of a high dose of E2 (OVX + high-E2). After 2 wk, rats were randomly assigned to coronary artery ligation (MI) and sham operation groups and studied after 3 wk. E2 status did not affect LV function in sham rats. At 2-3 wk after MI, impairment of LV function was similar across MI groups, as measured by echocardiography and direct LV catheterization. LV ACE mRNA abundance and activity were increased severalfold in all MI groups compared with respective sham animals and to similar levels across MI groups. In most brain nuclei, ACE and AT1R densities increased after MI. Unexpectedly, compared with the respective sham groups the relative increase was clearest (20-40%) in OVX + high-E2 MI rats, somewhat less (10-15%) in ovary-intact MI rats, and least (< 10-15%) in OVX + Veh MI rats. However, because in the sham group brain ACE and AT1R densities increased in the OVX + Veh rats and decreased in the OVX + high-E2 rats compared with the ovary-intact rats, actual ACE and AT1R densities in most brain nuclei were modestly higher (< 20%) in OVX + Veh MI rats than in the other two MI groups. Thus E2 does not inhibit upregulation of ACE in the LV after MI and amplifies the percent increases in ACE and AT1R densities in brain nuclei after MI, despite E2-induced downregulation in sham rats. Consistent with these minor variations in the tissue renin-angiotensin system, during the initial post-MI phase, E2 appears not to enhance or hinder the development of LV dysfunction.     

Serial magnetic resonance imaging based assessment of the early effects of an ACE inhibitor on postinfarction left ventricular remodeling in rats

In vivo assessment of treatment efficacy on postinfarct left ventricular (LV) remodeling is crucial for experimental studies. We examined the technical feasibility of serial magnetic resonance imaging (MRI) for monitoring early postinfarct remodeling in rats. MRI studies were performed with a 7-Tesla unit, 1, 3, 8, 15, and 30 days after myocardial infarction (MI) or sham operation, to measure LV mass, volume, and the ejection fraction (EF). Three groups of animals were analyzed: sham-operated rats (n = 6), MI rats receiving lisinopril (n = 11), and MI rats receiving placebo (n = 8). LV dilation occurred on day 3 in both MI groups. LV end-systolic and end-diastolic volumes were significantly lower in lisinopril-treated rats than in placebo-treated rats at days 15 and 30. EF was lower in both MI groups than in the sham group at all time points, and did not differ between the MI groups during follow-up. Less LV hypertrophy was observed in rats receiving lisinopril than in rats receiving placebo at days 15 and 30. We found acceptable within- and between-observer agreement and an excellent correlation between MRI and ex vivo LV mass (r = 0.96; p < 0.001). We demonstrated the ability of MRI to detect the early beneficial impact of angiotensin-converting enzyme (ACE) inhibitors on LV remodeling. Accurate and noninvasive, MRI is the tool of choice to document response to treatment targeting postinfarction LV remodeling in rats.     

Effects of captopril and omapatrilat on early post-myocardial infarction survival and cardiac hemodynamics in rats: interaction with cardiac cytokine expression

The purpose of this study was to evaluate and compare the effects of simultaneous angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) inhibition by the vasopeptidase inhibitor omapatrilat (10 and 40 mg x kg(-1) x day(-1)) with those of the selective ACE inhibitor captopril (160 mg x kg(-1) x day(-1)) on survival, cardiac hemodynamics, and cytokine mRNA expression in left ventricular (LV) tissues 4 days after myocardial infarction (MI) in rats. The effects of the co-administration of both B1 and B2 kinin receptor antagonists (2.5 mg x kg(-1) x day(-1) each) with and without omapatrilat were also evaluated to assess the role of bradykinin (BK) during this post-MI period. Both omapatrilat and captopril treatments improve early (4 days) post-MI survival when started 4 h post-MI. The use of kinin receptor antagonists had no significant effect on survival in untreated MI rats and omapatrilat-treated MI rats. This improvement in survival with omapatrilat and captopril is accompanied by a reduced LV end-diastolic pressure (LVEDP) and pulmonary congestion. The use of kinin receptor antagonists had little effect on cardiac hemodynamics or morphologic measurements. Acute MI significantly increased the expression of cardiac cytokines (TNF-alpha, TGF-beta1, and IL-10). Captopril significantly attenuated this activation, while omapatrilat had variable effects: sometimes increasing but generally not changing activation depending on the cytokine measured and the dose of omapatrilat used. The co-administration of both kinin receptor antagonists attenuates the increase in expression of cardiac TNF-alpha and TGF-beta1 after omapatrilat treatment. Taken together, these results would suggest that despite very marked differences in the way these drugs modified the expression of cardiac cytokines, both omapatrilat and captopril improved early (4 days) post-MI survival and cardiac function to a similar extent.     

Biventricular cardiac dysfunction after acute massive pulmonary embolism in the rat.

Cardiac dysfunction has been documented in vivo after acute massive pulmonary embolism (AMPE). The present study tests whether intrinsic ventricular dysfunction occurs in rat hearts isolated after AMPE. AMPE was induced in spontaneously breathing ketamine-xylazine-anesthetized rats by thrombus infusion until mean arterial blood pressure (MAP) was approximately 40% of basal measurement. A hypotensive control group underwent controlled blood withdrawal to produce MAP approximately 40% of basal levels. Shams underwent identical surgical and anesthesia preparation but without pulmonary embolization. Hearts were perfused in isovolumetric mode, and simultaneous right ventricular (RV) and left ventricular (LV) pressures were measured. AMPE caused arterial hypotension with hypoxemia (PO(2) = 50 +/- 14 Torr), acidemia (pH = 7.26 +/- 0.11), and high lactate concentration (6.9 +/- 1.7 mM). Starling curves from both ventricles demonstrated that AMPE significantly reduced ex vivo systolic contractile function in the RV (P = 0.031) and LV (P = 0.008) compared with both the hypotensive control and sham hearts. AMPE did not alter coronary flow or compliance in either ventricle. Soluble tumor necrosis factor-alpha decreased in the RV (P = 0.043) and LV (P = 0.005) tissue. These data support the hypothesis that AMPE produces intrinsic biventricular dysfunction and suggest that arterial hypotension is not the principal mechanism of this dysfunction.     

Regulation of cardiac and renal mineralocorticoid receptor expression by captopril following myocardial infarction in rats

Myocardial infarction (MI) activates the renin-angiotensin system in the heart and increases local production of aldosterone. This hormone may increase reactive fibrosis in the myocardium favoring heart failure development. To elucidate the potential contribution of aldosterone to cardiac remodeling following MI, we evaluated the expression of mineralocorticoid receptors (MCR) in the left ventricle (LV) and kidney of rats after MI and captopril treatment. MI was induced by ligation of the coronary artery in Wistar rats, which were separated into (1) sham-operated group, (2) MI group, (3) MI-captopril treated group (cap, 50 mg kg(-1) day(-1)). One month later angiotensin converting enzyme (ACE) activity was assayed in the plasma, LV and kidney. Cardiac and renal angiotensin II (Ang II) levels were determined by ELISA and MCR mRNA expression and protein were measured by Taqman RT-PCR and Western blot, respectively. Cardiac MCR mRNA and protein levels increased nearly by 80% after MI and Cap treatment normalized cardiac MCR protein and mRNA expression. Kidney MCR expression was not affected. ACE activity increased 34% in the plasma and 83% in the LV after MI. This increase was prevented by Cap. Ang II concentration increased 225% in the LV and 193% in kidney, which was partially attenuated by Cap. Our data demonstrate upregulation of MCR in the heart following MI what may facilitate the effects of aldosterone in the ventricular remodeling process. ACE inhibitors may reduce reactive fibrosis not only by decreasing Ang II production but also by attenuating the aldosterone-signaling pathway by decreasing the expression of MCR receptors.     

Susceptibility to systolic dysfunction in the myocardium from chronically infarcted spontaneously hypertensive rats

We explored whether the hypertensive heart is susceptible to myocardial dysfunction in viable noninfarcted tissue post-myocardial infarction (MI), the potential mechanisms thereof, and the impact of these changes on pump function. Six to seven months after the ligation of the left anterior descending coronary artery, left ventricular (LV) myocardial systolic function, as assessed from the percent shortening of the noninfarcted lateral wall segmental length determined over a range of filling pressures (ultrasonic transducers placed in the lateral wall in anaesthetized, open-chest, ventilated rats) and the percent thickening of the posterior wall (echocardiography), was reduced in infarcted spontaneous hypertensive rats (SHR-MI) (P < 0.05) but not in normotensive Wistar-Kyoto (WKY-MI) animals compared with corresponding controls [SHR-sham operations (Sham) and WKY-Sham]. This change in the regional myocardial function in SHR-MI, but not in WKY-MI, occurred despite a similar degree of LV dilatation (increased LV end-diastolic dimensions and volume intercept of the LV end-diastolic pressure-volume relation) in SHR-MI and WKY-MI rats and a lack of difference in LV relative wall thinning, LV wall stress, apoptosis [terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL)], or necrosis (pathological score) between SHR-MI and WKY-MI rats. Although the change in regional myocardial function in the SHR-MI group was not associated with a greater reduction in baseline global LV chamber systolic function [end-systolic elastance (LV E(es)) and endocardial fractional shortening determined in the absence of an adrenergic stimulus], in the presence of an isoproterenol challenge, noninfarct-zone LV systolic myocardial dysfunction manifested in a significant reduction in LV E(es) in SHR-MI compared with WKY-MI and SHR and WKY-Sham rats (P < 0.04). In conclusion, these data suggest that with chronic MI, the hypertensive heart is susceptible to the development of myocardial dysfunction, a change that cannot be attributed to excessive chamber dilatation, apoptosis, or necrosis, but which in turn contributes toward a reduced cardiac adrenergic inotropic reserve.     

Inhibition of matrix metalloproteinase activity by ACE inhibitors prevents left ventricular remodeling in a rat model of heart failure

Angiotensin-converting enzyme (ACE) inhibitors represent the front-line pharmacological treatment of heart failure, which is characterized by left ventricular (LV) dilatation and inappropriate hypertrophy. The mechanism of action of ACE inhibitors is still unclear, but evidence suggests that they may act by influencing matrix metalloproteinase (MMP) activity. This study sought to determine whether ACE inhibitors can directly regulate MMP activity and whether this results in positive structural and functional adaptations to the heart. To this end, MMP-2 activity in LV tissue extracted from rats with an aortocaval (AV) fistula was assessed by in vitro incubation as well as in vivo treatment with captopril, lisinopril, or quinapril. Furthermore, LV size and function were determined in untreated AV fistula rats, AV fistula rats treated with lisinopril (3, 5, and 8 wk), and age-matched sham-operated controls. In vitro incubation with captopril, lisinopril, or quinapril significantly reduced MMP-2 activity, as did in vivo treatment. This occurred without a reduction in the available pool of MMP-2 protein. Long-term in vivo administration of lisinopril also prevented LV dilatation, attenuated myocardial hypertrophy, and prevented changes in myocardial compliance and contractility. The results herein demonstrate that ACE inhibitors prevent MMP-2 activity and, in so doing, represent a mechanism responsible for preventing the negative structural and functional changes that occur in the rat AV fistula model of heart failure.     

Effects of the vasopeptidase inhibitor omapatrilat on cardiac endogenous kinins in rats with acute myocardial infarction

The purposes of this study were to evaluate and to compare the effects of simultaneous angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) inhibition by the vasopeptidase inhibitor omapatrilat (1 mg. kg(-1). day(-1)) with those of the selective ACE inhibitor enalapril (1 mg. kg(-1). day(-1)) on survival, cardiac hemodynamics, and bradykinin (BK) and des-Arg(9)-BK levels in cardiac tissues 24 h after myocardial infarction (MI) in rats. The effect of the co-administration of both B(1) and B(2) kinin receptor antagonists (2.5 mg. kg(-1). day(-1) each) with metallopeptidase inhibitors was also evaluated. The pharmacological treatments were infused subcutaneously using micro-osmotic pumps for 5 days starting 4 days before the ligation of the left coronary artery. Immunoreactive kinins were quantified by highly sensitive and specific competitive enzyme immunoassays. The post-MI mortality of untreated rats with a large MI was high; 74% of rats dying prior to the hemodynamic study. Mortality in the other MI groups was not significantly different from that of the untreated MI rats. Cardiac BK levels were not significantly different in the MI vehicle-treated group compared with the sham-operated rats. Both omapatrilat and enalapril treatments of MI rats significantly increased cardiac BK concentrations compared with the sham-operated group (P < 0.05). However, cardiac BK levels were significantly increased only in the MI omapatrilat-treated rats compared with the MI vehicle-treated group (P < 0.01). Cardiac des-Arg(9)-BK concentrations were not significantly modified by MI, and MI with omapatrilat or enalapril treatment compared with the sham-operated group. The co-administration of both kinin receptor antagonists with MI omapatrilat- and enalapril-treated rats had no significant effect on cardiac BK and des-Arg(9)-BK levels. Thus, the significant increase of cardiac BK concentrations by omapatrilat could be related to a biochemical or a cardiac hemodynamic parameter on early (24 h) post-MI state.     

β-Adrenergic receptors desensitization is not involved in exercise-induced cardiac fatigue: NADPH oxidase-induced oxidative stress as a new trigger

Prolonged strenuous exercise (PSE) induces transient left ventricular (LV) dysfunction. Previous studies suggest that β-adrenergic pathway desensitization could be involved in this phenomenon, but it remains to be confirmed. Moreover, other underlying mechanisms involving oxidative stress have been recently proposed. The present study aimed to evaluate the involvement of both the β-adrenergic pathway and NADPH oxidase (Nox) enzyme-induced oxidative stress in myocardial dysfunction in rats following PSE. Rats were divided into 4 groups: controls (Ctrl), 4-h exercised on treadmill (PSE), and 2 groups in which Nox enzyme was inhibited with apocynin treatment (Ctrl APO and PSE APO, respectively). We evaluated cardiac function in vivo and ex vivo during basal conditions and isoproterenol stress. GSH/GSSG ratio, cardiac troponin I (cTnI) release, and lipid peroxidation (MDA) were evaluated. PSE induced a decrease in LV developed pressure, intrinsic myocardial contractility, and relaxation associated with an increase in plasma cTnI release. Our in vivo and ex vivo results demonstrated no differences in myocardial response to isoproterenol and of effective dose 50 between control and PSE rats. Interestingly, the LV dysfunction was reversed by apocynin treatment. Moreover, apocynin prevented cellular oxidation [GSH/GSSG ratio: PSE APO rats vs. PSE rats in arbitrary units (au): 1.98 ± 0.07 vs. 1.35 ± 0.10; P < 0.001]. However, no differences in MDA were observed between groups. These data suggest that myocardial dysfunction observed after PSE was not due to β-adrenergic receptor desensitization but could be due to a signaling oxidative stress from the Nox enzyme.     

Age-related changes in cardiac structure and function in Fischer 344 x Brown Norway hybrid rats

The effects of aging on cardiovascular function and cardiac structure were determined in a rat model recommended for gerontological studies. A cross-sectional analysis assessed cardiac changes in male Fischer 344 x Brown Norway F1 hybrid rats (FBN) from adulthood to the very aged (n = 6 per 12-, 18-, 21-, 24-, 27-, 30-, 33-, 36-, and 39-mo-old group). Rats underwent echocardiographic and hemodynamic analyses to determine standard values for left ventricular (LV) mass, LV wall thickness, LV chamber diameter, heart rate, LV fractional shortening, mitral inflow velocity, LV relaxation time, and aortic/LV pressures. Histological analyses were used to assess LV fibrotic infiltration and cardiomyocyte volume density over time. Aged rats had an increased LV mass-to-body weight ratio and deteriorated systolic function. LV systolic pressure declined with age. Histological analysis demonstrated a gradual increase in fibrosis and a decrease in cardiomyocyte volume density with age. We conclude that, although significant physiological and morphological changes occurred in heart function and structure between 12 and 39 mo of age, these changes did not likely contribute to mortality. We report reference values for cardiac function and structure in adult FBN male rats through very old age at 3-mo intervals.     

Reduction of heart rate by chronic beta1-adrenoceptor blockade promotes growth of arterioles and preserves coronary perfusion reserve in postinfarcted heart

Adequate growth of coronary vasculature in the remaining left ventricular (LV) myocardium after myocardial infarction (post-MI) is a crucial factor for myocyte survival and performance. We previously demonstrated that post-MI coronary angiogenesis can be stimulated by bradycardia induced with the ATP-sensitive K(+) channel antagonist alinidine. In this study, we tested the hypothesis that heart rate reduction with beta-blockade may also induce coronary growth in the post-MI heart. Transmural MI was induced in 12-mo-old male Sprague-Dawley rats by occlusion of the left anterior descending coronary artery. Bradycardia was induced by administration of the beta-adrenoceptor blocker atenolol (AT) via drinking water (30 mg/day). Three groups of rats were compared: 1) control/sham (C/SH), 2) MI, and 3) MI + AT. In the MI + AT rats, heart rate was consistently reduced by 25-28% compared with C/SH rats. At 4 wk after left anterior descending coronary ligation, infarct size was similar in MI and MI + AT rats (67.1 and 61.5%, respectively), whereas a greater ventricular hypertrophy occurred in bradycardic rats, as indicated by a higher ventricular weight-to-body weight ratio (3.4 +/- 0.1 vs. 2.8 +/- 0.1 mg/g in MI rats). Analysis of LV function revealed a smaller drop in ejection fraction in the MI + AT than in the MI group ( approximately 24 vs. approximately 35%). Furthermore, in MI + AT rats, maximal coronary conductance and coronary perfusion reserve were significantly improved compared with the MI group. The better myocardial perfusion indexes in MI + AT rats were associated with a greater increase in arteriolar length density than in the MI group. Thus chronic reduction of heart rate induced with beta-selective blockade promotes growth of coronary arterioles and, thereby, facilitates regional myocardial perfusion in post-MI hearts.     

A new model of congestive heart failure in rats

Current rodent models of ischemia/infarct or pressure-volume overload are not fully representative of human heart failure. We developed a new model of congestive heart failure (CHF) with both ischemic and stress injuries combined with fibrosis in the remote myocardium. Sprague-Dawley male rats were used. Ascending aortic banding (Ab) was performed to induce hypertrophy. Two months post-Ab, ischemia-reperfusion (I/R) injury was induced by ligating the left anterior descending (LAD) artery for 30 min. Permanent LAD ligation served as positive controls. A debanding (DeAb) procedure was performed after Ab or Ab + I/R to restore left ventricular (LV) loading properties. Cardiac function was assessed by echocardiography and in vivo hemodynamic analysis. Myocardial infarction (MI) size and myocardial fibrosis were assessed. LV hypertrophy was observed 4 mo post-Ab; however, systolic function was preserved. LV hypertrophy regressed within 1 mo after DeAb. I/R for 2 mo induced a small to moderate MI with mild impairment of LV function. Permanent LAD ligation for 2 mo induced large MI and significant cardiac dysfunction. Ab for 2 mo followed by I/R for 2 mo (Ab + I/R) resulted in moderate MI with significantly reduced ejection fraction (EF). DeAb post Ab + I/R to reduce afterload could not restore cardiac function. Perivascular fibrosis in remote myocardium after Ab + I/R + DeAb was associated with decreased cardiac function. We conclude that Ab plus I/R injury with aortic DeAb represents a novel model of CHF with increased fibrosis in remote myocardium. This model will allow the investigation of vascular and fibrotic mechanisms in CHF characterized by low EF, dilated LV, moderate infarction, near-normal aortic diameter, and reperfused coronary arteries.     

Aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation

It is well established that the aging heart exhibits left ventricular (LV) diastolic dysfunction and changes in mechanical properties, which are thought to be due to alterations in the extracellular matrix. We tested the hypothesis that the mechanical properties of cardiac myocytes significantly change with aging, which could contribute to the global changes in LV diastolic dysfunction. We used atomic force microscopy (AFM), which determines cellular mechanical property changes at nanoscale resolution in myocytes, from young (4 mo) and old (30 mo) male Fischer 344 x Brown Norway F1 hybrid rats. A measure of stiffness, i.e., apparent elastic modulus, was determined by analyzing the relationship between AFM indentation force and depth with the classical infinitesimal strain theory and by modeling the AFM probe as a blunted conical indenter. This is the first study to demonstrate a significant increase (P < 0.01) in the apparent elastic modulus of single, aging cardiac myocytes (from 35.1 +/- 0.7, n = 53, to 42.5 +/- 1.0 kPa, n = 58), supporting the novel concept that the mechanism mediating LV diastolic dysfunction in aging hearts resides, in part, at the level of the myocyte.     

Cytochrome P-450 metabolite of arachidonic acid mediates bradykinin-induced negative inotropic effect

This study focused on the mechanisms of the negative inotropic response to bradykinin (BK) in isolated rat hearts perfused at constant flow. BK (100 nM) significantly reduced developed left ventricular pressure (LVP) and the maximal derivative of systolic LVP by 20-22%. The cytochrome P-450 (CYP) inhibitors 1-aminobenzotriazole (1 mM and 100 microM) or proadifen (5 microM) abolished the cardiodepression by BK, which was not affected by nitric oxide and cyclooxygenase inhibitors (35 microM NG-nitro-L-arginine methyl ester and 10 microM indomethacin, respectively). The CYP metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET; 50 ng/ml) produced effects similar to those of BK in terms of the reduction in contractility. After the coronary endothelium was made dysfunctional by Triton X-100 (0.5 microl), the BK-induced negative inotropic effect was completely abolished, whereas the 14,15-EET-induced cardiodepression was not affected. In hearts with normal endothelium, after recovery from 14,15-EET effects, BK reduced developed LVP to a 35% greater extent than BK in the control. In conclusion, CYP inhibition or endothelial dysfunction prevents BK from causing cardiodepression, suggesting that, in the rat heart, endothelial CYP products mediate the negative inotropic effect of BK. One of these mediators appears to be 14,15-EET.     

Kallikrein activates bradykinin B2 receptors in absence of kininogen

Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.     

Losartan prevents contractile dysfunction in rat myocardium after left ventricular myocardial infarction

We studied the effects of chronic losartan (Los) treatment on contractile function of isolated right ventricular (RV) trabeculae from rat hearts 12 wk after left ventricular (LV) myocardial infarction (MI) had been induced by ligation of the left anterior descending artery at 4 wk of age. After recovery, one-half of the animals were started on Los treatment (MI+Los; 30 mg x kg(-1) x day(-1) per os); the remaining animals were not treated (MI group). Rats without infarction or Los treatment served as controls (Con group). MI resulted in increases in LV and RV weight and unstressed LV cavity diameter; these were partially prevented by Los treatment. The active peak twitch force-sarcomere length relation was depressed in MI compared with either Con or MI+Los. Likewise, maximum Ca2+ saturated twitch force was depressed in MI, whereas twitch relaxation and twitch duration were prolonged. Myofilament function, as measured in skinned trabeculae, was not significantly different among the Con, MI, and MI+Los groups. We conclude that Los prevents contractile dysfunction in rat RV trabeculae after LV MI. Our results suggest that the beneficiary effect of Los treatment results not from improved myofilament function but rather from improved myocyte Ca2+ homeostasis.