Brian R. Bond

A Matrix Metalloproteinase Induction/Activation System Exists in the Human Left Ventricular Myocardium and Is Upregulated in Heart Failure

BackgroundMatrix metalloproteinases (MMPs) contribute to matrix remodeling in disease states such as tumor metastases. Extracellular matrix metalloproteinase inducer (EMMPRIN) has been reported to increase MMP expression, and membrane-type MMP or MT1-MMP has been implicated to activate MMPs. The present study examined whether and to what degree EMMPRIN and MT1-MMP were expressed in human left ventricular (LV) myocardium as well as the association with MMP activity and expression in dilated cardiomyopathy (DCM). Methods and ResultsLV myocardial zymographic MMP activity increased by >2-fold with both nonischemic DCM (n=21) and ischemic DCM (n=16) compared with normal (n=13). LV myocardial abundance of MMP-9 was increased with both forms of DCM. MMP-2 and MMP-3 were increased with nonischemic DCM. MMP-1 levels were decreased with both forms of DCM. EMMPRIN increased by >250% and MT1-MMP increased by >1000% with both forms of DCM. ConclusionsIncreased LV myocardial MMP activity and selective upregulation of MMPs with nonischemic and ischemic forms of DCM occurred. Moreover, a local MMP induction/activation system was identified in isolated normal human LV myocytes that was upregulated with DCM. The control of MMP activation and expression in the failing human LV myocardium represents a new and potentially significant therapeutic target for this disease process.

Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target.

A fundamental structural event in the progression of heart failure due to dilated cardiomyopathy is left ventricular (LV) myocardial remodeling. The matrix metalloproteinases (MMPs) are an endogenous family of enzymes which contribute to matrix remodeling in several disease states. The goal of this report is to summarize recent findings regarding the myocardial MMP system and the relation to matrix remodeling in the failing heart. In both experimental and clinical forms of dilated cardiomyopathy (DCM), increased expression of certain species of myocardial MMPs have been demonstrated. Specifically, increased myocardial levels of the gelatinase, MMP-9 has been identified in both ischemic and non-ischemic forms of human DCM. In addition, stromelysin or MMP-3 increased by over four-fold in DCM. The increased levels of MMP-3 in DCM may have particular importance since this MMP degrades a wide range of extracellular proteins and can activate other MMPs. In normal human LV myocardium, the membrane type 1 MMP (MT1-MMP) was detected. These MT-MMPs may provide important sites for local MMP activation within the myocardium. In a pacing model of LV failure, MMP expression and activity increased early and were temporally associated with LV myocardial matrix remodeling. Using a broad-spectrum pharmacological MMP inhibitor in this pacing model, the degree of LV dilation was attenuated and associated with an improvement in LV pump function. Thus, increased LV myocardial MMP expression and activity are contributory factors in the LV remodeling process in cardiomyopathic disease states. Regulation of myocardial MMP expression and activity may be an important therapeutic target for controlling myocardial matrix remodeling in the setting of developing heart failure.

Magnesium supplementation in the prevention of arrhythmias in pediatric patients undergoing surgery for congenital heart defects

Abstract Background The efficacy of magnesium in the prevention of arrhythmias in pediatric patients after heart surgery remains unknown. Therefore we prospectively examined the effect of magnesium treatment on the incidence of postoperative arrhythmias in pediatric patients undergoing surgical repair of congenital heart defects. Methods and Results Twenty-eight pediatric patients undergoing heart surgery with cardiopulmonary bypass were prospectively, randomly assigned in a double-blind fashion to receive intravenous magnesium (magnesium group, n=13; 30 mg/kg) or saline (placebo group, n=15) immediately after cessation of cardiopulmonary bypass. Magnesium, potassium, and calcium levels were measured at defined intervals during surgery and 24 hours after surgery. Continuous electrocardiographic documentation by Halter monitor was performed for 24 hours after surgery. Magnesium levels were significantly decreased below the normal reference range for patients in the placebo group compared with the magnesium group on arrival in the intensive care unit and for 20 hours after surgery. Magnesium levels remained in the normal range for patients in the magnesium group after magnesium supplementation. In 4 patients in the placebo group (27%), junctional ectopic tachycardia developed within the initial 20 hours in the intensive care unit. No junctional ectopic tachycardia was observed in the magnesium group ( P = .026). Conclusions Although this study was originally targeted to include 100 patients, the protocol was terminated because of the unacceptable incidence of hemodynamically significant junctional ectopic tachycardia that was present in the placebo group. Thus low magnesium levels in pediatric patients undergoing heart surgery are associated with an increased incidence of junctional ectopic tachycardia in the immediate postoperative period.

Sensitization of Human Atrial 5-HT4 Receptors by Chronic β-Blocker Treatment

Background Chronic treatment of patients with β-blockers induces β2-adrenergic receptor hyperresponsiveness in atrium and sinoatrial node. To investigate whether other atrial Gs protein–coupled receptors also become hyperresponsive after chronic treatment with β-blockers, we investigated 5-HT4 receptors in tissues and myocytes, which mediate serotonin-evoked increases of both contractile force and cAMP levels. Methods and Results Isolated right atrial strips from patients who had been chronically treated or not treated with a β-blocker were set up to contract. In tissues from β-blocker–treated patients (n=27), the maximum inotropic response to serotonin was 56±3% (mean±SEM) of the effect elicited by (−)-isoproterenol (200 μmol/L) compared with a response of 19±6% in tissues from non–β-blocker–treated patients (n=13) (P<.001). The responsiveness of the tissues to Ca2+ was unchanged by chronic β-blocker treatment. Serotonin (1 and 10 μmol/L) increased tissue cAMP levels, the increase with 10 μmol/L being si...

Differential effects of calcium channel antagonists in the amelioration of radial artery vasospasm

BACKGROUND Radial artery (RA) is being used for coronary artery bypass grafting (CABG) with greater frequency. However, RA is prone to post-CABG vasospasm, which may be neurohormonally mediated. Use of the calcium channel antagonist diltiazem has been advocated as a strategy to reduce post-CABG RA vasospasm. However, whether and to what degree different calcium channel antagonists influence neurohormonally induced RA vasoconstriction remains unknown. METHODS RA segments were collected from patients undergoing elective CABG (n = 13), and isometric tension was examined in the presence of endothelin (10 nM) or norepinephrine (1 microM). In matched RA, endothelin- or norepinephrine-induced contractions were measured in the presence of diltiazem (277 nM), amlodipine (73 nM), or nifedipine (145 nM). These concentrations of calcium channel antagonists were based upon clinical plasma profiles. RESULTS Endothelin and norepinephrine caused a significant increase in RA-developed tension (0.54+/-0.1 and 0.68+/-0.1 g/mg, respectively; p<0.05). Amlodipine or nifedipine significantly reduced RA vasoconstriction in the presence of endothelin (30+/-6% and 41+/-9%, respectively; p<0.05) or norepinephrine (27+/-8% and 53+/-9%, respectively; p<0.05), whereas diltiazem did not significantly reduce RA vasoconstriction. CONCLUSIONS These results demonstrate that neurohormonal factors released post-CABG can cause RA vasoconstriction, and that calcium channel antagonists are not equally effective in abrogating that response. Both amlodipine and nifedipine, which have a higher degree of vascular selectivity, appear to be the most effective in reducing RA vasoconstriction.

Endothelin receptor pathway in human left ventricular myocytes: relation to contractility

BACKGROUND Increased synthesis and release of the potent bioactive peptide endothelin-1 (ET-1) occurs during and after cardiac surgery. However, the cellular and molecular basis for the effects of ET-1 on human left ventricular (LV) myocyte contractility remains unknown. METHODS LV myocyte contractility was examined from myocardial biopsies taken from patients (n = 30) undergoing elective coronary artery bypass. LV myocytes (n = 997, > 30/patient) were isolated using microtrituration and contractility examined by videomicroscopy at baseline and after ET-1 exposure (200 pmol/L). In additional studies, myocytes were pretreated to inhibit either protein kinase C (PKC) (chelerythrine, 1 micromol/L), the sodium/hydrogen (Na/H) exchanger (EIPA, 1 micromol/L), both PKC and the Na/H exchanger, or the ET(A) receptor (BQ-123, 1 micromol/L), followed with ET-1 exposure. RESULTS Basal myocyte shortening increased 37.8 +/- 6.3% with ET-1 (p < 0.05). Na/H exchanger, PKC, and dual inhibition all eliminated the effects of ET-1. Furthermore, ET(A) inhibition demonstrated that ET-1 effects on myocyte contractility were mediated through the ET(A) receptor subtype. CONCLUSIONS ET-1 directly influences human LV myocyte contractility, which is mediated through the ET(A) receptor and requires intracellular activation of PKC and stimulation of the Na/H exchanger.

Renal Organic Anion Transporter-Mediated Drug-Drug Interaction between Gemcabene and Quinapril

In humans and rats, a synergistic blood pressure reduction was observed when the fibrate gemcabene (CI-1027) was coadministered with the angiotensin-converting enzyme inhibitor quinapril. In a quinapril (3 mg/kg) pharmacokinetic rat study, there was a 40% decrease in urinary excretion and a 53% increase in plasma area under the curve from 0 to 24 h of the active metabolite quinaprilat when coadministered with gemcabene (30 mg/kg). This observation revealed a possible transporter-mediated drug-drug interaction (DDI) between gemcabene and quinapril. This led to a series of studies investigating the underlying clearance mechanisms associated with these compounds intended to elucidate renal transporter interactions between quinapril and gemcabene. In vitro transporter studies using human embryonic kidney 293 cells transfected with human or rat organic anion transporter 3 (hOAT3, rOat3) revealed that quinaprilat is a substrate in both species, with a Km value of 13.4 μM for hOAT3. Subsequent studies discovered that gemcabene inhibited quinaprilat uptake by hOAT3 and rOat3 at IC50 values of 35 and 48 μM, respectively. Moreover, gemcabene acylglucuronide, the major metabolite of gemcabene glucuronidation, also inhibited hOAT3- and rOat3-mediated uptake of quinaprilat at IC50 values of 197 and 133 μM, respectively. High plasma concentrations of gemcabene (>100 μM) achieved in humans and rats upon oral dosing corroborate with gemcabene inhibition of renal OAT3-mediated secretion of quinaprilat in vitro. This investigation established that a DDI between gemcabene and quinapril involving inhibition of renal transporters and subsequent elevation in plasma concentrations of quinaprilat is responsible for the apparent synergistic blood pressure reduction observed with these compounds.

Identification of mRNA transcripts and immunohistochemical localization of Na/H exchanger isoforms in gerbil inner ear

Recent physiological and pharmacological studies have implicated involvement of the Na/H exchanger (NHE) in regulating inner ear ion homeostasis, but the cellular distribution of this membrane transporter remains unknown. Here reverse transcription and the polymerase chain reaction (RT-PCR) were employed to screen adult gerbil inner ears for mRNA transcripts encoding the four best characterized isoforms of NHE. PCR products spanning selected segments of NHE mRNAs were cloned and sequenced. The putative housekeeping gene NHE-1 was found to be expressed and the 459 bp product shared 98.7% amino acid homology with rat sequence. NHE-2, NHE-3 and NHE-4 cDNA transcripts likewise were detected and the PCR products shared 100, 99.4 and 88.9% amino acid homology, respectively, with their rat counterparts. In addition, the cellular distribution of NHE isoforms 1 and 3 was mapped in the gerbil inner ear by immunostaining with polyclonal antisera against rat antigens. In the cochlea, the antiserum against NHE-1 reacted strongly at the basolateral membrane of strial marginal cells as well as with inner and outer hair cells and spiral ganglion neurons. Less intense staining for NHE-1 was present in subpopulations of fibrocytes in the spiral limbus and in inferior and superior areas of the spiral ligament. In the vestibular system dark and transitional cells expressed abundant NHE-1 as did hair cells and vestibular ganglia neurons. Immunostaining with the antiserum against NHE-3 was limited to the apical surface of marginal cells in the stria vascularis. Based on these data, NHE-1 likely functions primarily to maintain intracellular pH levels in cells where it is found in high abundance. NHE-3, on the other hand, possibly participates in the vectorial transcellular movement of Na+ by strial marginal cells thus helping to maintain the extremely low Na+ level in cochlear endolymph.

Myocyte endothelin exposure during cardioplegic arrest exacerbates contractile dysfunction after reperfusion.

Transient left ventricular (LV) dysfunction can occur after cardioplegic arrest. The contributory mechanisms for this phenomenon are not completely understood. We tested the hypothesis that exposure of LV myocytes to endothelin (ET) during simulated cardioplegic arrest would have direct effects on contractile processes with subsequent reperfusion. LV porcine myocytes were randomly assigned to three groups: 1) Control: normothermic (37°C) cell media (n = 204); 2) Cardioplegia: simulated cardioplegic arrest (K+ 24 mEq/L, 4°C × 2 h) followed by reperfusion and rewarming with cell media (n = 164); and 3) Cardioplegia/ ET simulated cardioplegic arrest in the presence of ET (200 pM) followed by reperfusion with cell media containing ET (n = 171). Myocyte contractility was measured by computer-assisted video microscopy. In a subset of experiments, myocyte intracellular calcium was determined after Fluo-3 (Molecular Probes, Eugene, OR) loading by digital fluorescence image analysis. Myocyte shortening velocity was reduced after cardioplegic arrest compared with controls (52 ± 2 vs 84 ± 3 &mgr;m/s, respectively;P < 0.05) and was further reduced with cardioplegic arrest and ET exposure (43 ± 2 &mgr;m/s, P < 0.05). Intracellular calcium was significantly increased in myocytes exposed to cardioplegia compared with normothermic control myocytes and was further augmented by cardioplegia with ET supplementation (P < 0.05). Exposure of the LV myocyte to ET during cardioplegic arrest directly contributed to contractile dysfunction after reperfusion. Moreover, alterations in intracellular calcium may play a role in potentiatiing the myocyte contractile dysfunction associated with ET exposure during cardioplegic arrest. Implications Exposure of the left ventricular myocyte to endothelin during cardioplegic arrest directly contributed to contractile dysfunction after reperfusion. Moreover, alterations in intracellular calcium may play a role in potentiating the myocyte contractile dysfunction associated with endothelin exposure during cardioplegic arrest.

Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea.

Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil–treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy.