Selective action of metoprolol to attenuate regadenoson-induced tachycardia in conscious dogs.

BACKGROUND: Regadenoson is a coronary vasodilator that causes tachycardia via activation of the sympathetic nervous system. We determined whether ß(1)-adrenergic blockade can attenuate tachycardia without significantly reducing coronary vasodilation induced by regadenoson. METHODS AND RESULTS: Hemodynamics and coronary blood flow (CBF) were measured in conscious dogs. Baseline CBF and heart rate (HR) were 42 ± 2 mL/min and 87 ± 8 bpm (mean ± SEM), respectively. Regadenoson (1, 2.5, and 5 µg/kg) increased peak CBF by 129 ± 10, 149 ± 7, and 174 ± 10 mL/min and HR by 48 ± 6, 67 ± 5, and 85 ± 11 bpm, respectively, (all P < .05 vs baseline). In the presence of metoprolol (1.5 mg/kg), the peak increases in CBF caused by these three doses of regadenoson were reduced by only 11 ± 7%, 10 ± 4%, and 21 ± 2% (P = NS, <.05, and <.05 vs regadenoson alone), respectively, whereas the regadenoson-induced tachycardia was significantly reduced by 55 ± 8%, 55 ± 4%, and 52 ± 5% (all P < .05). In the presence of metoprolol, the duration of the regadenoson-induced increase in CBF was reduced, but the duration of the 2-fold increase in CBF caused by 5 µg/kg regadenoson was still nearly 6 minutes. CONCLUSION: ß(1)-Adrenergic blockade with metoprolol attenuated the regadenoson-induced increase in HR more than the increase in CBF.

Intramyocardial VEGF-B167 gene delivery delays the progression towards congestive failure in dogs with pacing-induced dilated cardiomyopathy.

RATIONALE: Vascular endothelial growth factor (VEGF)-B selectively binds VEGF receptor (VEGFR)-1, a receptor that does not mediate angiogenesis, and is emerging as a major cytoprotective factor. OBJECTIVE: To test the hypothesis that VEGF-B exerts non-angiogenesis-related cardioprotective effects in nonischemic dilated cardiomyopathy. METHODS AND RESULTS: AAV-9-carried VEGF-B(167) cDNA (10(12) genome copies) was injected into the myocardium of chronically instrumented dogs developing tachypacing-induced dilated cardiomyopathy. After 4 weeks of pacing, green fluorescent protein-transduced dogs (AAV-control, n=8) were in overt congestive heart failure, whereas the VEGF-B-transduced (AAV-VEGF-B, n=8) were still in a well-compensated state, with physiological arterial Po(2). Left ventricular (LV) end-diastolic pressure in AAV-VEGF-B and AAV-control was, respectively, 15.0+/-1.5 versus 26.7+/-1.8 mm Hg and LV regional fractional shortening was 9.4+/-1.6% versus 3.0+/-0.6% (all P<0.05). VEGF-B prevented LV wall thinning but did not induce cardiac hypertrophy and did not affect the density of alpha-smooth muscle actin-positive microvessels, whereas it normalized TUNEL-positive cardiomyocytes and caspase-9 and -3 activation. Consistently, activated Akt, a major negative regulator of apoptosis, was superphysiological in AAV-VEGF-B, whereas the proapoptotic intracellular mediators glycogen synthase kinase (GSK)-3beta and FoxO3a (Akt targets) were activated in AAV-control, but not in AAV-VEGF-B. Cardiac VEGFR-1 expression was reduced 4-fold in all paced dogs, suggesting that exogenous VEGF-B(167) exerted a compensatory receptor stimulation. The cytoprotective effects of VEGF-B(167) were further elucidated in cultured rat neonatal cardiomyocytes exposed to 10(-8) mol/L angiotensin II: VEGF-B(167) prevented oxidative stress, loss of mitochondrial membrane potential, and, consequently, apoptosis. CONCLUSIONS: We determined a novel, angiogenesis-unrelated cardioprotective effect of VEGF-B(167) in nonischemic dilated cardiomyopathy, which limits apoptotic cell loss and delays the progression toward failure.

Identification of a coronary vascular progenitor cell in the human heart.

Primitive cells capable of generating small resistance arterioles and capillary structures in the injured myocardium have been identified repeatedly. However, these cells do not form large conductive coronary arteries that would have important implications in the management of the ischemic heart. In the current study, we determined whether the human heart possesses a class of progenitor cells that regulates the growth of endothelial cells (ECs) and smooth muscle cells (SMCs) and vasculogenesis. The expression of vascular endothelial growth-factor receptor 2 (KDR) was used, together with the stem cell antigen c-kit, to isolate and expand a resident coronary vascular progenitor cell (VPC) from human myocardial samples. Structurally, vascular niches composed of c-kit-KDR-positive VPCs were identified within the walls of coronary vessels. The VPCs were connected by gap junctions to ECs, SMCs, and fibroblasts that operate as supporting cells. In vitro, VPCs were self-renewing and clonogenic and differentiated predominantly into ECs and SMCs and partly into cardiomyocytes. To establish the functional import of VPCs, a critical stenosis was created in immunosuppressed dogs, and tagged human VPCs were injected in proximity to the constricted artery. One month later, there was an increase in coronary blood flow (CBF) distal to the stenotic artery, resulting in functional improvement of the ischemic myocardium. Regenerated large, intermediate, and small human coronary arteries and capillaries were found. In conclusion, the human heart contains a pool of VPCs that can be implemented clinically to form functionally competent coronary vessels and improve CBF in patients with ischemic cardiomyopathy.

Peroxide generation by p47phox-Src activation of Nox2 has a key role in protein kinase C-induced arterial smooth muscle contraction.

Protein kinase C (PKC) stimulation of NAD(P)H oxidases (Nox) is an important component of multiple vascular disease processes; however, the relationship between oxidase activation and the regulation of vascular smooth muscle contraction by PKC remains poorly understood. Therefore, we examined the signaling cascade of PKC-elicited Nox activation and the role of superoxide and hydrogen peroxide in mediating PKC-induced vascular contraction. Endothelium-denuded bovine coronary arteries showed a PKC-dependent basal production of lucigenin (5 muM)-detected Nox oxidase-derived superoxide, which was stimulated fourfold by PKC activation with 10 muM phorbol 12,13-dibutyrate (PDBu). PDBu appeared to increase superoxide generation by Nox2 through both p47(phox) and peroxide-dependent Src activation mechanisms based on the actions of inhibitors, properties of Src phosphorylation, and the loss of responses in aorta from mice deficient in Nox2 and p47(phox). The actions of inhibitors of contractile regulating mechanisms, scavengers of superoxide and peroxide, and responses in knockout mouse aortas suggest that a major component of the contraction elicited by PDBu appeared to be mediated through peroxide derived from Nox2 activation stimulating force generation through Rho kinase and calmodulin kinase-II mechanisms. Superoxide generated by PDBu also attenuated relaxation to nitroglycerin. Peroxide-derived from Nox2 activation by PKC appeared to be a major contributor to the thromboxane A(2) receptor agonist U46619 (100 nM)-elicited contraction of coronary arteries. Thus a p47(phox) and Src kinase activation of peroxide production by Nox2 appears to be an important contributor to vascular contractile mechanisms mediated through activation of PKC.

Regadenoson, a novel pharmacologic stress agent for use in myocardial perfusion imaging, does not have a direct effect on the QT interval in conscious dogs.

Our goal was to determine the effect of regadenoson (a novel A2A adenosine receptor agonist) on the QT interval in conscious dogs. Sixteen mongrel dogs were chronically instrumented for measurements of blood pressure and ECG. Regadenoson (2.5, 5, and 10 microg/kg, IV) caused a dose-dependent QT interval shortening (DeltaQT: 14 +/- 3, 24 +/- 5, and 27 +/- 5 ms, mean +/- SEM; n = 7 to 11; all P < 0.05) associated with significant increases in HR (Peak HR: 114 +/- 9, 125 +/- 6, and 144 +/- 7 bpm). Atrial pacing (135, 150, and 165 bpm) also caused a frequency-dependent shortening of the QT interval (DeltaQT: 15 +/- 3, 22 +/- 3, and 39 +/- 5 ms; n = 6 to 7; all P < 0.05). Regadenoson- and pacing-induced shortenings in the QT interval were significantly correlated with the R-R interval (r = 0.67 and 0.8, both P < 0.05). Regadenoson at 5 and 10 microg/kg did not cause a significant change in HR or QT interval either during atrial pacing at 165 bpm or after administration of propranolol and atropine to prevent HR from changing or after treatment of dogs with hexamethonium to block autonomic ganglia. Regadenoson (5 to 10 microg/kg) caused no significant changes of QT interval in the heart in which HR was kept constant via physiological or pharmacological procedures, indicating that regadenoson has no direct effect on the QT interval.

Coronary nitric oxide production controls cardiac substrate metabolism during pregnancy in the dog.

The aim of this study was to examine the role of nitric oxide (NO) in the control of cardiac metabolism at 60 days of pregnancy (P60) in the dog. There was a basal increase in diastolic coronary blood flow during pregnancy and a statistically significant increase in cardiac output (55 +/- 4%) and in cardiac NOx production (44 +/- 4 to 59 +/- 3 nmol/min, P < 0.05). Immunohistochemistry of the left ventricle showed an increase in endothelial nitric oxide synthase staining in the endothelial cells at P60. NO-dependent coronary vasodilation (Bezold-Jarisch reflex) was increased by 20% and blocked by N(G)-nitro-l-arginine methyl ester (l-NAME). Isotopically labeled substrates were infused to measure oleate, glucose uptake, and oxidation. Glucose oxidation was not significantly different in P60 hearts (5.4 +/- 0.5 vs. 6.2 +/- 0.4 micromol/min) but greatly increased in response to l-NAME injection (to 19.9 +/- 0.9 micromol/min, P < 0.05). Free fatty acid (FFA) oxidation was increased in P60 (from 5.3 +/- 0.6 to 10.4 +/- 0.5 micromol/min, P < 0.05) and decreased in response to l-NAME (to 4.5 +/- 0.5 micromol/min, P < 0.05). There was an increased oxidation of FFA for ATP production but no change in the respiratory quotient during pregnancy. Genes associated with glucose and glycogen metabolism were downregulated, whereas genes involved in FFA oxidation were elevated. The acute inhibition of NO shifts the heart away from FFA and toward glucose metabolism despite the downregulation of the carbohydrate oxidative pathway. The increase in endothelium-derived NO during pregnancy results in a tonic inhibition of glucose oxidation and reliance on FFA uptake and oxidation to support ATP synthesis in conjunction with upregulation of FFA metabolic enzymes.

Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan.

Heart failure is the leading cause of death in the elderly, but whether this is the result of a primary aging myopathy dictated by depletion of the cardiac progenitor cell (CPC) pool is unknown. Similarly, whether current lifespan reflects the ineluctable genetic clock or heart failure interferes with the genetically determined fate of the organ and organism is an important question. We have identified that chronological age leads to telomeric shortening in CPCs, which by necessity generate a differentiated progeny that rapidly acquires the senescent phenotype conditioning organ aging. CPC aging is mediated by attenuation of the insulin-like growth factor-1/insulin-like growth factor-1 receptor and hepatocyte growth factor/c-Met systems, which do not counteract any longer the CPC renin-angiotensin system, resulting in cellular senescence, growth arrest, and apoptosis. However, pulse-chase 5-bromodeoxyuridine-labeling assay revealed that the senescent heart contains functionally competent CPCs that have the properties of stem cells. This subset of telomerase-competent CPCs have long telomeres and, following activation, migrate to the regions of damage, where they generate a population of young cardiomyocytes, reversing partly the aging myopathy. The senescent heart phenotype and heart failure are corrected to some extent, leading to prolongation of maximum lifespan.

Caffeine attenuates the duration of coronary vasodilation and changes in hemodynamics induced by regadenoson (CVT-3146), a novel adenosine A2A receptor agonist.

Effects of caffeine on regadenoson-induced coronary vasodilation and changes in hemodynamics were examined in conscious dogs. Sixteen dogs were chronically instrumented for measurements of coronary blood flow (CBF), mean arterial pressure (MAP), and heart rate (HR). Regadenoson (5 microg/kg, IV) increased CBF from 34 +/- 2 to 191 +/- 7 mL/min. The duration of the 2-fold increase in CBF was 515 +/- 71 seconds. Regadenoson decreased MAP by 15 +/- 2% and increased HR by 114 +/- 14%. Regadenoson-induced maximum increases in CBF were not significantly lower in the presence of caffeine at 1, 2, 4, and 10 mg/kg (2 +/- 3, 0.7 +/- 3, 16 +/- 5, and 13 +/- 8%, respectively; all P > 0.05). Caffeine at 1, 2, 4, and 10 mg/kg significantly decreased the duration of the 2-fold increase in CBF induced by regadenoson by 17% +/- 4%, 48% +/- 8%, 62% +/- 5%, and 82% +/- 5%, respectively (all P < 0.05). Caffeine at 4 and 10 mg/kg significantly attenuated the effects of regadenoson on MAP and HR. The results indicate that 1 to 10 mg/kg caffeine dose-dependently reduced the duration, but not the peak increase of CBF caused by 5 microg/kg regadenoson.

Adeno-associated virus-mediated transduction of VEGF165 improves cardiac tissue viability and functional recovery after permanent coronary occlusion in conscious dogs.

We have previously shown that VEGF165 gene delivery into ischemic skeletal muscle exerts not only proangiogenic, but also remarkable antiapoptotic and proregenerative activity. The aim of this study was to determine whether recombinant adeno-associated virus (rAAV)-mediated gene delivery of VEGF165 into cardiac muscle, during acute myocardial infarction, exerts a protective effect to promote long-term functional recovery. Acute infarction of the anterior LV wall was induced in 12 chronically instrumented dogs by permanent occlusion of the LAD coronary artery. Four hours after occlusion, rAAV-VEGF165 or rAAV-LacZ (n=6 each; 5x10(12) viral particles per animal) was directly injected with an echo-guided needle into the dysfunctional cardiac wall. LV and arterial pressure, dP/dtmax, and ejection fraction were not significantly different between the two groups over time. In contrast, in the infarcted region, at four weeks after infarction, fractional shortening was 75+/-18% and -3+/-15% of baseline and length-pressure area was 54+/-15% and 0.8+/-15% of baseline in VEGF165 versus LacZ, respectively (P<0.05). Histological analysis of the border regions showed a marked increase in the number of alpha-SMA-positive arterioles (68+/-2.8 versus 100+/-3.8 vessels per microscopic field in LacZ and VEGF165 group, respectively; P<0.05). In both groups, the receptor VEGFR-2 was diffusely expressed on the surviving cardiomyocytes and, consistently, myocardial viability was significantly improved in the VEGF165-treated group, with several troponin T-expressing cardiomyocytes displaying nuclear positivity for the proliferation marker PCNA. Altogether, our results indicate that VEGF165 gene delivery exerts a marked beneficial action by enhancing both arteriologenesis and cardiomyocyte viability in infarcted myocardium.

Endothelial dysfunction and hypertension in rats transduced with CYP4A2 adenovirus.

Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114+/-1 to 133+/-1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2-transduced rats produced more 20-HETE (129+/-10 versus 97+/-7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2+/-1.6 versus 8.4+/-1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.

cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation.

The objective of this study was to determine whether activation of protein kinase B (PKB) is involved in the production of nitric oxide (NO) induced by cAMP signal transduction. Mongrel dogs were used for this study. Coronary microvessels were isolated from the left ventricular free wall of these dog hearts. Forskolin (an activator of adenylyl cyclase that increases intracellular cAMP level) and 8-bromo-cAMP (a membrane-permeable cAMP analog) were used to stimulate NO release and activation of PKB and endothelial NO synthase (eNOS) in these blood vessels. We found that forskolin and 8-bromo-cAMP increased NO release (quantified by using the Griess reaction) from coronary microvessels by 80 +/- 6 and 78 +/- 11 pmol/mg (mean +/- SE), respectively (P < 0.05 vs. control). Western blot analysis showed that forskolin elicited a significant increase in eNOS phosphorylation (59 +/- 11%) at serine-1177 (a positively regulatory phosphorylation site for eNOS) and a significant increase in dephosphorylation (28 +/- 6%) at threonine-495 (a negatively regulatory phosphorylation site of eNOS) (P < 0.05 vs. control). Interestingly, forskolin also increased the phosphorylation of PKB at serine-473 (by 49 +/- 17%) and threonine-308 (by 53 +/- 17%), respectively (P < 0.05 vs. control; phosphorylation of both sites is required for a full activation of PKB). N(omega)-nitro-l-arginine methyl ester (an NOS inhibitor) blocked NO formation, Rp diastereomer of cAMP (a PKA inhibitor), and LY-294002 [a PI3-kinase (an activator of PKB) inhibitor] prevented the production of NO, phosphorylation of PKB, and eNOS induced by forskolin. Our data clearly show an involvement of PKB activation in cAMP signal-induced NO production. We are reporting for the first time that cAMP signal transduction stimulates eNOS activation through a PKB-mediated mechanism.

Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function.

The purpose of this study was to determine whether the heart in large mammals contains cardiac progenitor cells that regulate organ homeostasis and regenerate dead myocardium after infarction. We report that the dog heart possesses a cardiac stem cell pool characterized by undifferentiated cells that are self-renewing, clonogenic, and multipotent. These clonogenic cells and early committed progeny possess a hepatocyte growth factor (HGF)-c-Met and an insulin-like growth factor 1 (IGF-1)-IGF-1 receptor system that can be activated to induce their migration, proliferation, and survival. Therefore, myocardial infarction was induced in chronically instrumented dogs implanted with sonomicrometric crystals in the region of the left ventricular wall supplied by the occluded left anterior descending coronary artery. After infarction, HGF and IGF-1 were injected intramyocardially to stimulate resident cardiac progenitor cells. This intervention led to the formation of myocytes and coronary vessels within the infarct. Newly generated myocytes expressed nuclear and cytoplasmic proteins specific of cardiomyocytes: MEF2C was detected in the nucleus, whereas alpha-sarcomeric actin, cardiac myosin heavy chain, troponin I, and alpha-actinin were identified in the cytoplasm. Connexin 43 and N-cadherin were also present. Myocardial reconstitution resulted in a marked recovery of contractile performance of the infarcted heart. In conclusion, the activation of resident primitive cells in the damaged dog heart can promote a significant restoration of dead tissue, which is paralleled by a progressive improvement in cardiac function. These results suggest that strategies capable of activating the growth reserve of the myocardium may be important in cardiac repair after ischemic injury.

Exogenous nitric oxide reduces glucose transporters translocation and lactate production in ischemic myocardium in vivo.

Nitric oxide (NO) inhibits myocardial glucose transport and metabolism, although the underlying mechanism(s) and functional consequences of this effect are not clearly understood. We tested the hypothesis that NO inhibits the activation of AMP-activated protein kinase (AMPK) and translocation of cardiac glucose transporters (GLUTs; GLUT-4) and reduces lactate production. Ischemia was induced in open-chest dogs by a 66% flow reduction in the left anterior descending coronary artery (LAD). During ischemia, dogs were untreated (control) or treated by direct LAD infusion of (i) nitroglycerin (NTG) (0.5 microg.kg(-1).min(-1)); (ii) 8-Br-cGMP (50 microg.kg(-1).min(-1)); or (iii) NO synthase inhibitor L-nitro-argininemethylester (40 microg.kg(-1).min(-1); n = 9 per group). Cardiac substrate oxidation was measured with isotopic tracers. There were no differences in myocardial blood flow or oxygen delivery among groups; however, at 45 min of ischemia, the activation of AMPK was significantly less in NTG (77 +/- 12% vs. nonischemic myocardium) and 8-Br-cGMP (104 +/- 13%), compared with control (167 +/- 17%). Similarly, GLUT-4 translocation was significantly reduced in NTG (74 +/- 7%) and 8-Br-cGMP (120 +/- 11%), compared with control (165 +/- 17%). Glucose uptake and lactate output were 30% and 60% lower in NTG compared with control. Inhibition of NO synthesis stimulated glucose oxidation (67% increase compared with control) but did not affect AMPK phosphorylation, GLUT-4 translocation and glucose uptake. Contractile function in the ischemic region was significantly improved by NTG and L-nitro-argininemethylester. In conclusion, in ischemic myocardium an NO donor inhibits glucose uptake and lactate production via a reduction in AMPK stimulation of GLUT-4 translocation, revealing a mechanism of metabolic modulation and myocardial protection activated by NO donors.

gp91phox-containing NAD(P)H oxidase mediates attenuation of nitric oxide-dependent control of myocardial oxygen consumption by ANG II.

We have previously reported that ANG II stimulation increased superoxide anion (O2-) through the activation of NAD(P)H oxidase and inhibited nitric oxide (NO)-dependent control of myocardial oxygen consumption (MVo2) by scavenging NO. Our objective was to investigate the role of NAD(P)H oxidase, especially the gp91phox subunit, in the NO-dependent control of MVo2. MVo2 in mice with defects in the expression of gp91phox [gp91(phox)(-/-)] was measured with a Clark-type oxygen electrode. Baseline MVo2 was not significantly different between wild-type (WT) and gp91(phox)(-/-) mice. Stimulation of NO production by bradykinin (BK) induced significant decreases in MVo2 in WT mice. BK-induced reduction in MVo2 was enhanced in gp91(phox)(-/-) mice. BK-induced reduction in MVo2 in WT mice was attenuated by 10(-8) mol/l ANG II, which was restored by coincubation with Tiron or apocynin. In contrast to WT mice, BK-induced reduction in MVo2 in gp91(phox)(-/-) mice was not altered by ANG II. There was a decrease in lucigenin (5 x 10(-6) mol/l)-detectable O2- in gp91(phox)(-/-) mice compared with WT mice. ANG II resulted in significant increases in O2- production in WT mice, which was inhibited by coincubation with Tiron or apocynin. However, ANG II had no effect on O2- production in gp91(phox)(-/-) mice. Histological examination showed that the development of abscesses and/or the invasion of inflammatory cells occurred in lungs and livers but not in hearts and kidneys from gp91(phox)(-/-) mice. These results indicate that the gp91(phox) subunit of NAD(P)H oxidase mediates O2- production through the activation of NAD(P)H oxidase and attenuation of NO-dependent control of MVo2 by ANG II.

Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice.

The role of mitochondrial manganese-superoxide dismutase (Mn-SOD) in the maintenance of vascular function has not yet been studied. Thus we examined flow- and agonist-induced dilations in isolated mesenteric arteries (approximately 90 microm in diameter) of Mn-SOD heterozygous (Mn-SOD+/-) and wild-type (WT) mice. Increases in flow elicited dilations in all vessels, but the magnitude of the dilation was significantly less in vessels of Mn-SOD+/- mice than in those of WT mice (64 vs. 74% of passive diameter). N(omega)-nitro-L-arginine methyl ester inhibited the dilation in vessels of WT mice but had no effect on vessels of Mn-SOD+/- mice. Tempol or tiron (superoxide scavengers) increased flow-induced dilation in vessels of Mn-SOD+/- mice. Acetylcholine- and sodium nitroprusside-induced, but not adenosine-induced, dilations were also decreased in arteries of Mn-SOD+/- mice. Superoxide levels in the arteries of Mn-SOD+/- mice were significantly increased. Western blot analysis confirmed a 50% reduction of Mn-SOD protein in the vessels of Mn-SOD+/- mice. A 41% reduction in endothelial nitric oxide synthase (eNOS) protein and a 37% reduction in eNOS activity were also found in the vessels of Mn-SOD+/- mice. Whereas there was no difference in eNOS protein in kidney homogenates of WT and Mn-SOD+/- mice, a significant reduction of nitric oxide synthase activity was found in Mn-SOD+/- mice, which could be restored by the administration of tiron. We conclude that an increased concentration of superoxide due to reduced activity of Mn-SOD, which inactivates nitric oxide and inhibits eNOS activity, contributes to the impaired vasodilator function of isolated mesenteric arteries of Mn-SOD+/- mice. These results suggest that Mn-SOD contributes significantly to the regulation of vascular function.

Paradoxical downregulation of the glucose oxidation pathway despite enhanced flux in severe heart failure.

Free fatty acid (FFA) oxidation is depressed in severe heart failure due to reduced activity of mitochondrial fatty acid oxidation enzymes. It is unknown whether the concomitant enhancement in cardiac glucose use is a consequence of reduced FFA oxidation, or also due to potentiation of the carbohydrate oxidative pathway. FFA and glucose oxidation rates were measured in vivo in 9 normal dogs and 9 dogs with pacing-induced heart failure by infusing (3)H-oleate and (14)C-glucose. FFA oxidation was lower (39 +/- 9 vs. 73 +/- 5 nmol min(-1) g(-1)), while glucose oxidation was higher (42 +/- 8 vs. 17 +/- 6 nmol min(-1) g(-1)) in failing compared to normal hearts (P < 0.05). At the end of the in vivo experiment, clamp-frozen biopsies were harvested from the left ventricle. Messenger RNAs encoding for proteins involved in both glucose and fatty acid metabolism, and for citrate synthase, were significantly reduced. Protein expression of GLUT-1 and GLUT-4, and GLUT-4 translocation to the sarcolemma showed no significant differences between the two groups despite a significant reduction in mRNAs with heart failure. GAPDH mRNA, protein expression, and activity were all reduced. The E2 subunit of pyruvate dehydrogenase was decreased both at the mRNA and protein level, with no effect on either fractional or maximal activity. In conclusion, we found either no changes or moderate downregulation of key enzymes of the carbohydrate metabolism in failing hearts, which suggests that the increase in glucose oxidation in vivo was principally due to impaired FFA oxidation and that the maximal myocardial capacity to obtain energy from substrate is globally depressed.

Comparative profile of vasodilation by CVT-3146, a novel A2A receptor agonist, and adenosine in conscious dogs.

The purpose of this study was to determine the magnitude of vasodilation by CVT-3146 in different vascular beds and to compare it with that by adenosine in conscious dogs. Intravenous bolus injections of CVT-3146 (0.1-2.5 microg/kg) or adenosine (10-250 microg/kg) caused a dose-dependent increase in the coronary blood flow (CBF) and a dose-dependent decrease in the late diastolic coronary resistance. Although the maximal increase in CBF response to the two drugs was not significantly different, the ED50 of CVT-3146 and adenosine were 0.45 +/- 0.07 microg/kg and 47 +/- 7.77 microg/kg, respectively. The highest dose of CVT-3146 caused a much longer coronary vasodilation than the highest dose of adenosine. There were no significant differences in increases in cardiac output induced by higher doses of CVT-3146 or adenosine. Most importantly, CVT-3146 resulted in a smaller decrease in total peripheral resistance (TPR) compared to that seen with adenosine. In addition, CVT-3146 yielded a smaller increase in the lower body flow (LBF) than adenosine. Adenosine also caused dose-dependent renal vasoconstriction, whereas CVT-3146 did not affect the renal blood flow. The administration of CVT-3146 or adenosine caused a dose-dependent vasodilation in the mesentery, which was not significantly different from each other. In summary, CVT-3146 is a 100-fold more potent coronary vasodilator than adenosine. CVT-3146 causes smaller decreases in TPR and smaller increases in LBF than those induced by adenosine, indicating that it is more selective for coronary than peripheral vasodilation. Furthermore, CVT-3146 did not cause renal vasoconstriction. These features make CVT-3146 a better candidate for pharmacologic stress testing.

NAD(P)H oxidase-generated superoxide anion accounts for reduced control of myocardial O2 consumption by NO in old Fischer 344 rats.

We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in Fischer 344 rats. In Fischer rats at 4, 14, and 23 mo of age, we examined cardiac function using echocardiography, the regulation of cardiac O2 consumption in vitro, endothelial NO synthase (eNOS) protein levels, and potential mechanisms that regulate superoxide. Aging was associated with a reduced ejection fraction [from 75 +/- 2% at 4 mo to 66 +/- 3% (P < 0.05) at 23 mo] and an increased cardiac diastolic volume [from 0.60 +/- 0.04 to 1.00 +/- 0.10 ml (P < 0.01)] and heart weight (from 0.70 +/- 0.02 to 0.90 +/- 0.02 g). The NO-mediated control of cardiac O2 consumption by bradykinin or enalaprilat was not different between 4 mo (36 +/- 2 or 34 +/- 3%) and 14 mo (29 +/- 1 or 25 +/- 3%) but markedly (P < 0.05) reduced in 23-mo-old Fischer rats (15 +/- 3 or 7 +/- 2%). The response to the NO donor S-nitroso-N-acetyl penicillamine was not different across groups (35%, 35%, and 44%). Interestingly, the eNOS protein level was not different at 4, 14, and 23 mo. The addition of tempol (1 mmol/l) to the tissue bath eliminated the depression in the control of cardiac O2 consumption by bradykinin (25 +/- 3%) or enalaprilat (28 +/- 3%) in 23-mo-old Fischer rats. We next examined the levels of enzymes involved in the production and breakdown of superoxide. The expression of Mn SOD, Cu/Zn SOD, extracellular SOD, and p67phox, however, did not differ between 4- and 23-mo-old rats. Importantly, there was a marked increase in gp91phox, and apocynin restored the defect in NO-dependent control of cardiac O2 consumption at 23 mo to that seen in 4-mo-old rats, identifying the role of NADPH oxidase. Thus increased biological activity of superoxide and not decreases in the enzyme that produces NO are responsible for the altered control of cardiac O2 consumption by NO in 23-mo-old Fischer rats. Increased oxidant stress in aging, by decreasing NO bioavailability, may contribute not only to changes in myocardial function but also to altered regulation of vascular tone and the progression of cardiac or vascular disease.

Adeno-associated virus mediated gene delivery into coronary microvessels of chronically instrumented dogs.

The objective of this study was to assess the potential of adeno-associated virus (AAV)-mediated gene delivery into coronary microvessels in vivo in a large animal. Ten mongrel dogs were chronically instrumented and allowed to recover for 10 days. Dogs were reanesthetized, and the aorta was constricted by a hydraulic occluder, whereby left ventricular (LV) pressure increased by 30% and left circumflex coronary artery blood flow by 50%. Recombinant AAV (serotype 2, CMV enhancer/chicken beta-actin promoter) encoding for green fluorescent protein (GFP) was injected as a bolus into the left atrium during aortic constriction at total titers of 1010 or 1012 infectious units. Dogs were followed for 2 (n = 4)or4wk(n = 6). Hemodynamics or body weight did not change. In LV tissue slices, a fluorescein-labeled antibody to GFP stained endothelial and smooth muscle cells but was absent in myocytes. To quantify transduction, slices were then stained with antibodies against alpha-smooth muscle actin or von Willebrand factor. Approximately 4% of arterioles and 2% of microvessels stained positive for anti-GFP independent from viral titer or duration. By regression analyses, the percent of vessels transfected was proportional to the increase in LV systolic pressure during occlusion. AAV is a potential vector for gene transfer into the coronary microcirculation in large animals, including perhaps humans.

Selective A2A adenosine receptor agonist as a coronary vasodilator in conscious dogs: potential for use in myocardial perfusion imaging.

The authors sought to demonstrate the advantages of a selective, potent, short-acting A adenosine receptor agonist, CVT-3146 (2-(N-pyrazolyl)Ado derivative), for potential clinical use as a coronary vasodilator during myocardial perfusion imaging. The use of adenosine in a pharmacological stress test during myocardial imaging is limited by side effects mediated by A1 and A2B adenosine receptors and by its ultrashort duration of action. CVT-3146 (0.1-5 microg/kg) and adenosine (13-267 microg/kg) were given as peripheral intravenous injections in 10 awake dogs instrumented for measurement of coronary blood flow (CBF). CVT-3146 caused a dose-dependent increase of CBF (ED50 = 0.34 +/- 0.08 microg/kg, maximal increase = 221 +/- 18%, n = 6). Adenosine was less potent (ED = 51 +/- 15 microg/kg, p < 0.05) but equieffective (maximal increase in CBF = 227 +/- 11%). The increase in CBF caused by 2.5 microg/kg CVT-3146 reached 84 +/- 5% of the maximal reactive hyperemia following 20 s of coronary occlusion (n = 4). After a 10-s injection of CVT-3146 (2.5 microg/kg), the increase in CBF remained at least twofold above baseline for 97 +/- 14 s, whereas for adenosine (267 microg/kg), the twofold increase in CBF lasted only 24 +/- 2 s (p < 0.01, n = 6). A 30-s injection of 2.5 microg/kg CVT-3146 prolonged the twofold increase in CBF up to 221 +/- 20 s. No atrioventricular block was noted. At 2.5 microg/kg, the peak effect of CVT-3146 on CBF was associated with a short-lasting (20 +/- 6 s) increase in heart rate (78 +/- 9 bpm) and decrease in mean arterial blood pressure (13 +/- 6 mm Hg, p < 0.05, n = 6). CVT-3146 is a potent coronary vasodilator. Its short duration of action, minimal and transient systemic hemodynamic effects, and ease of administration may make this agonist suitable for pharmacological coronary vasodilation during myocardial perfusion imaging for noninvasive detection of subcritical arterial stenosis.