The eNOS enhancer AVE 9488: a novel cardioprotectant against ischemia reperfusion injury.

Nitric oxide (NO) is an important regulator of vascular and myocardial function. Cardiac ischemia/reperfusion injury is reduced in mice overexpressing endothelial NO synthase (eNOS) suggesting cardioprotection by eNOS. Novel pharmacological substances, so called eNOS enhancers, upregulate eNOS expression and thereby increase NO production. We tested the effects of the eNOS enhancer AVE 9488 on cardiac ischemia/reperfusion injury in vivo in mice. After treatment with the eNOS enhancer AVE 9488 (30 mg/kg/day) or placebo for one week mice underwent 30 min of coronary artery ligation and 24 h of reperfusion in vivo. Ischemia-reperfusion damage was significantly reduced in mice treated with the eNOS enhancer when compared to placebo treated mice (infarct/area at risk 65.4 +/- 4.1 vs. 36.9 +/- 4.0%, placebo vs. eNOS enhancer, P = 0.0002). The protective effect was blunted in eNOS knockout mice treated with the eNOS enhancer (infarct/area at risk 64.1 +/- 6.2%, eNOS knockout + eNOS enhancer vs. WT + eNOS enhancer, P = ns). Reactive oxygen species were significantly reduced in mice treated with the eNOS enhancer as indicated by significantly lower malondialdehyde-thiobarbituric acid levels (placebo vs. eNOS enhancer, 3.2 +/- 0.5 vs. 0.8 +/- 0.07 micromol/l, P = 0.0003). Thus pharmacological interventions addressed to increase eNOS-derived NO production constitute a promising therapeutic approach to prevent myocardial ischemia/reperfusion injury.

Dobutamine-stress magnetic resonance microimaging in mice : acute changes of cardiac geometry and function in normal and failing murine hearts.

The aim of this study was to assess the capability of MRI to characterize systolic and diastolic function in normal and chronically failing mouse hearts in vivo at rest and during inotropic stimulation. Applying an ECG-gated FLASH-cine sequence, MRI at 7 T was performed at rest and after administration of 1.5 microgram/g IP dobutamine. There was a significant increase of heart rate, cardiac output, and ejection fraction and significant decrease of end-diastolic and end-systolic left ventricular (LV) volumes (P<0.01 each) in normal mice during inotropic stimulation. In mice with heart failure due to chronic myocardial infarction (MI), MRI at rest revealed gross LV dilatation. There was a significant decrease of LV ejection fraction in infarcted mice (29%) versus sham mice (58%). Mice with MI showed a significantly reduced maximum LV ejection rate (P<0.001) and LV filling rate (P<0.01) and no increase of LV dynamics during dobutamine action, indicating loss of contractile and relaxation reserve. In 4-month-old transgenic mice with cardiospecific overexpression of the beta(1)-adrenergic receptor, which at this early stage do not show abnormalities of resting cardiac function, LV filling rate failed to increase after dobutamine stress (transgenic, 0.19+/-0.03 microL/ms; wild type, 0.36+/-0.01 microL/ms; P<0.01). Thus, MRI unmasked diastolic dysfunction during dobutamine stress. Dobutamine-stress MRI allows noninvasive assessment of systolic and diastolic components of heart failure. This study shows that MRI can demonstrate loss of inotropic and lusitropic response in mice with MI and can unmask diastolic dysfunction as an early sign of cardiac dysfunction in a transgenic mouse model of heart failure.

Chronic phosphocreatine depletion by the creatine analogue beta-guanidinopropionate is associated with increased mortality and loss of ATP in rats after myocardial infarction.

BACKGROUND: The failing myocardium is characterized by reductions of phosphocreatine (PCr) and free creatine content and by decreases of energy reserve via creatine kinase (CK), ie, CK reaction velocity (Flux(CK)). It has remained unclear whether these changes contribute directly to contractile dysfunction. In the present study, myocardial PCr stores in a heart failure model were further depleted by feeding of the PCr analogue beta-guanidinopropionate (GP). Functional and metabolic consequences were studied. METHODS AND RESULTS: Rats were subjected to sham operation or left coronary artery ligation (MI). Surviving rats were assigned to 4 groups and fed with 0% (n=7, Sham; n=5, MI) or 1% (n=7 Sham+GP, n=8 MI+GP) GP. Two additional groups were fed GP for 2 or 4 weeks before MI. After 8 weeks, hearts were isolated and perfused, and left ventricular pressure-volume curves were obtained. High-energy phosphate metabolism was determined with (31)P NMR spectroscopy. After GP feeding or MI, left ventricular pressure-volume curves were depressed by 33% and 32%, respectively, but GP feeding in MI hearts did not further impair mechanical function. Both MI and GP feeding reduced PCr content and Flux(CK), but here, effects were additive. In MI+GP rats, PCr levels and Flux(CK) were reduced by 87% and 94%, respectively. Although ATP levels were maintained in the GP and MI groups, ATP content was reduced by 18% in MI+GP hearts. Furthermore, 24-hour mortality in GP-prefed rats was 100%. CONCLUSIONS: Rats with an 87% predepletion of myocardial PCr content cannot survive an acute MI. Chronically infarcted hearts subjected to additional PCr depletion cannot maintain ATP homeostasis.

Evidence against a role of physiological concentrations of estrogen in post-myocardial infarction remodeling.

OBJECTIVES: The purpose of this study was to examine whether endogenous estrogen deficiency induced by ovariectomy affects chronic left ventricular dysfunction post-myocardial infarction (MI). BACKGROUND: Epidemiologic findings suggest that mortality of postmenopausal women is increased after MI, but the underlying mechanisms are unknown. METHODS: Rats were either not ovariectomized (non-OVX), ovariectomized (OVX) or ovariectomized and treated with subcutaneous 17-beta-estradiol (E2) pellets (OVX + E2). Two weeks later, animals were sham-operated (Sham) or left coronary artery ligated (MI). Eight weeks later, in vivo echocardiographic and hemodynamic measurements were performed. Thereafter, hearts were isolated and perfused isovolumically. RESULTS: Mean infarct size was similar among the three MI groups. Ovariectomy decreased serum E2 levels (11 +/- 4 vs. 49 +/- 11 pg/ml in non-OVX, p < 0.01) and increased body weight. These changes were reversed by E2 replacement. The degree of cardiac hypertrophy was similar for all groups post-MI. Left ventricular diameters were increased post-MI (8.9 +/- 0.4 in non-OVX + MI vs. 6.7 +/- 0.2 mm in non-OVX + Sham hearts, p < 0.0001), but OVX or OVX + E2 replacement did not alter left ventricular diameters in post-MI and Sham hearts. Left ventricular fractional shortening was severely impaired post-MI (19 +/- 2% vs. 50 +/- 3 in non-OVX + Sham hearts, p < 0.0001) with no influence of hormonal status. Left ventricular end-diastolic pressure, measured in vivo, was increased in all MI groups without significant differences between groups. Pressure-volume curves, obtained in perfused hearts, demonstrated a right and downward shift with reduced maximum left ventricular developed pressure post-MI (75 +/- 6 vs. 108 +/- 3 mm Hg in non-OVX + Sham hearts, p < 0.001) and were also unaffected by either OVX or E2 replacement. CONCLUSIONS: Chronic endogenous estrogen deficiency does not have major effects on the development of cardiac hypertrophy, dysfunction and dilation post-MI.

Chronic high-dose creatine feeding does not attenuate left ventricular remodeling in rat hearts post-myocardial infarction.

OBJECTIVE: In heart failure, cardiac energy metabolism is compromised. The failing myocardium is characterized by reduced contents of both phosphorylated (phosphocreatine) and non-phosphorylated (free) creatine content as well as decreased energy reserve via creatine kinase (creatine kinase reaction velocity). These changes may contribute to cardiac dysfunction. The purpose of the present study was to determine whether chronic feeding with high-dose dietary creatine prevents the derangement of energy metabolism and the development of left ventricular remodeling in a rat model of heart failure, i.e. post-myocardial infarction (MI). METHODS AND RESULTS: Rats were subjected to sham operation or left coronary artery ligation. Surviving rats were fed with 0% (untreated) or 3% creatine (related to weight of diet) for 8 weeks. Creatine feeding increased serum creatine levels significantly approximately 2-fold. Thereafter, hearts were isolated, perfused and left ventricular pressure-volume curves obtained. Steady state and dynamic (CK reaction velocity) high-energy phosphate metabolism was determined with 31P NMR spectroscopy. In both MI groups (treated n = 8, untreated n = 7), pressure-volume curves were shifted right- and downward compared to both sham groups (treated n = 5, untreated n = 7), i.e. creatine had no effect on left ventricular remodeling. Likewise, similar reductions of phosphocreatine, free creatine and creatine kinase reaction velocity (untreated sham 12.0 +/- 0.7 mmol/lxs; untreated MI 7.8 +/- 0.7*; treated sham 13.6 +/- 1.0; treated MI 7.2 +/- 1.1*; *p < 0.025 sham vs. MI) were found in both MI groups. CONCLUSIONS: Chronic creatine feeding of post-MI rats is ineffective in preventing the functional and energetic derangements occurring post-MI. Inspite of increased serum creatine levels, neither the normal nor the failing heart accumulates additional creatine.

Preservation of cardiac function and energy reserve by the angiotensin-converting enzyme inhibitor quinapril during postmyocardial infarction remodeling in the rat.

PURPOSE: Angiotensin-converting enzyme (ACE) inhibitors show beneficial long-term hemodynamic effects in chronically infarcted hearts. The purpose of this study was to test whether prevention of the deterioration of mechanical function by ACE inhibitors is related to beneficial effects on high-energy phosphate metabolism that is deranged in heart failure. METHODS: Twelve-week old rats were randomly assigned to ligation of the left coronary artery [mycardial infarction (MI)] or sham operation (Sham) and to the ACE inhibitor quinapril (+Q) (6 mg/kg/day per gavage) or placebo treatment. Eight weeks later, cardiac function was measured in the isolated heart by a left ventricular balloon (pressure-volume curves), and energy metabolism of residual intact myocardium was analyzed in terms of total and isoenzyme creatine kinase activity (spectrophotometry), steady-state levels [adenosine triptosphate (ATP), phosphocreatine], and turnover rates (creatine kinase reaction velocity) of high-energy phosphates [31P nuclear magnetic resonance (NMR)] and total creatine content [high-performance liquid chromatography (HPLC)]. RESULTS: Quinapril prevented post-MI hypertrophy and partially prevented left ventricular contractile dysfunction [maximum left ventricular developed pressure 166+/-6, 83+/-16 (p < 0.05 MI vs. Sham), 139+/-13 mm Hg (p < 0.05 quinapril treated vs. untreated) in Sham, MI and MI+Q hearts]. Residual intact failing myocardium showed a 17% decrease of MM-CK and a 16% decrease of mito-CK activity. Total creatine was reduced by 23%, phosphocreatine by 26% and CK reaction velocity by 30%. Parallel to improved function, treatment with quinapril largely prevented the impairment of energy metabolism occuring post-MI. CONCLUSIONS: quinapril treatment results in an improvement of high-energy phosphate metabolism, of energy reserve via the creatine kinase reaction, and of contractile performance post-MI.

Effects of ACE inhibition and beta-receptor blockade on energy metabolism in rats postmyocardial infarction.

Chronic treatment with beta-receptor blockers or angiotensin-converting enzyme (ACE) inhibitors in heart failure can reduce mortality and improve left ventricular function, but the mechanisms involved in their beneficial action remain to be fully defined. Our hypothesis was that these agents prevent the derangement of cardiac energy metabolism. Rats were subjected to myocardial infarction (MI) or sham operation. Thereafter, animals were treated with bisoprolol, captopril, or remained untreated. Two months later, cardiac function was measured in the isolated heart by a left ventricular balloon (pressure-volume curves), and energy metabolism of residual intact myocardium was analyzed in terms of total and isoenzyme creatine kinase (CK) activity, steady-state levels (ATP, phosphocreatine), and turnover rates (CK reaction velocity) of high-energy phosphates (31P nuclear magnetic resonance) and total creatine content (HPLC). Bisoprolol and partially captopril prevented post-MI hypertrophy and partially prevented left ventricular contractile dysfunction. Residual intact failing myocardium in untreated, infarcted hearts showed a 25% decrease of the total, a 26% decrease of MM-, and a 37% decrease of the mitochondrial CK activity. Total creatine was reduced by 15%, phosphocreatine by 21%, and CK reaction velocity by 41%. Treatment with bisoprolol or captopril largely prevented all of these changes in infarcted hearts. Thus the favorable functional effects of beta-receptor blockers and ACE inhibitors post-MI are accompanied by substantial beneficial effects on cardiac energy metabolism.

Detection of myocardial viability based on measurement of sodium content: A (23)Na-NMR study.

MRI of total sodium (Na) content may allow assessment of myocardial viability, but information on Na content in normal myocardium, necrotic/scar tissue, and stunned or hibernating myocardium is lacking. Thus, the aims of the study were to: 1) quantify the temporal changes in myocardial Na content post-myocardial infarction (MI) in a rat model (Protocol 1); 2) compare Na in normally perfused, hibernating, and stunned canine myocardium (Protocol 2); and 3) determine whether, in buffer-perfused rat hearts, infarct scar can be differentiated from intact myocardium by (23)Na-MRI (Protocol 3). In Protocol 1, rats were subjected to LAD ligation. Infarct/scar tissue was excised at control and 1, 3, 7, 28, 56, and 128 days post-MI (N = 6-8 each), Na content was determined by (23)Na-NMR spectroscopy (MRS) and ion chromatography. Na content was persistently increased at all time points post-MI averaging 306*-160*% of control values (*P < 0.0083 vs. control). In Protocol 2, (23)Na-MRS of control (baseline), stunned and hibernating samples revealed no difference in Na. In Protocol 3, (23)Na-MRI revealed a mean increase in signal intensity, to 142 +/- 6% of control values, in scar tissue. A threshold of 2 standard deviations of the image intensity allowed determination of infarct size, correlating with histologically determined infarct size (r = 0.91, P < 0.0001).