Increased inducible nitric oxide synthase and arginase II expression in heart failure: no net nitrite/nitrate production and protein S-nitrosylation.

Our objective was to address the balance of inducible nitric oxide (NO) synthase (iNOS) and arginase and their contribution to contractile dysfunction in heart failure (HF). Excessive NO formation is thought to contribute to contractile dysfunction; in macrophages, increased iNOS expression is associated with increased arginase expression, which competes with iNOS for arginine. With substrate limitation, iNOS may become uncoupled and produce reactive oxygen species (ROS). In rabbits, HF was induced by left ventricular (LV) pacing (400 beats/min) for 3 wk. iNOS mRNA [quantitative real-time PCR (qRT-PCR)] and protein expression (confocal microscopy) were detected, and arginase II expression was quantified with Western blot; serum arginine and myocardial nitrite and nitrate concentrations were determined by chemiluminescence, and protein S-nitrosylation with Western blot. Superoxide anions were quantified with dihydroethidine staining. HF rabbits had increased LV end-diastolic diameter [20.0 + or - 0.5 (SE) vs. 17.2 + or - 0.3 mm in sham] and decreased systolic fractional shortening (11.1 + or - 1.4 vs. 30.6 + or - 0.7% in sham; both P < 0.05). Myocardial iNOS mRNA and protein expression were increased, however, not associated with increased myocardial nitrite or nitrate concentrations or protein S-nitrosylation. The serum arginine concentration was decreased (124.3 + or - 5.6 vs. 155.4 + or - 12.0 micromol/l in sham; P < 0.05) at a time when cardiac arginase II expression was increased (0.06 + or - 0.01 vs. 0.02 + or - 0.01 arbitrary units in sham; P < 0.05). Inhibition of iNOS with 1400W attenuated superoxide anion formation and contractile dysfunction in failing hearts. Concomitant increases in iNOS and arginase expression result in unchanged NO species and protein S-nitrosylation; with substrate limitation, uncoupled iNOS produces superoxide anions and contributes to contractile dysfunction.

New insights into the S-nitrosothiol-ascorbate reaction. The formation of nitroxyl.

Although the ascorbate-dependent reduction of S-nitrosothiol to the corresponding thiol function is frequently used for analyzing S-nitrosocysteinyl residues in proteins, it proceeds with low yields. Our re-investigation of the ascorbate-S-nitrosothiol reaction demonstrates now the intermediacy of nitroxyl (HNO/3NO-) that is highly effective in oxidizing thiols. The occurrence of the HNO reporter molecule, i.e., N2O, during and after reaction was unequivocally demonstrated by 15N NMR. The yield of HNO from the S-nitrosoglutathione-ascorbate reaction was determined with the aid of a Mn(III)-complex to 60%, a value that was significantly higher than the one of nitric oxide formation (48%) at physiological pH. The same HNO yield was observed with a S-nitrosothiol bound to a protein (i.e., S-nitroso-papain). With the known chemistry of nitroxyl, it was possible to optimize the experimental conditions so that the GSNO-ascorbate reaction yielded stoichiometric amounts of glutathione after a reaction period of 1 min.

MR-compatible polyetheretherketone-based guide wire assisting MR-guided stenting of iliac and supraaortic arteries in swine: feasibility study.

The purpose of this study was to demonstrate first magnetic resonance (MR)-guided stenting of iliac and supraaortic arteries using a polyetheretherketone-based (PEEK) MR-compatible guide wire. In vitro and animal experiments were performed in a short magnet wide-bore scanner (1.5 Tesla, Espree, Siemens Healthcare, Erlangen, Germany). For all experiments, a 0.035'' MR-compatible guide wire prototoype was used. This wire had a compound core of PEEK with reinforcing fibres, a soft and atraumatic tip and a hydrophilic coating. For its passive visualization, paramagnetic markings were attached. All experiments were performed through a vascular introducer sheath under MR-guidance. In vitro repetitive selective over the wire catheterizations of either the right carotid artery and the left subclavian artery were performed. In vivo, selective catheterization and over-the-wire stenting of the brachiocephalic trunk and the left subclavian artery were performed. The common iliac arteries were catheterized retrogradely (left) and cross-over (right). Angioplasty and stenting were performed over-the-wire. All procedures were successful. Visibility of the PEEK-based guide-wire was rated good in vitro and acceptable in vivo. Guide wire pushability and endovascular device support were good. The PEEK-based MR-compatible guide wire is well visible and usable under MR-guidance. It supports over-the-wire treatment of iliac arteries and supraaortic arteries.

Feasibility of real-time magnetic resonance-guided angioplasty and stenting of renal arteries in vitro and in Swine, using a new polyetheretherketone-based magnetic resonance-compatible guidewire.

OBJECTIVES: Demonstrate the usability of a new polyetheretherketone (PEEK)-based MR-compatible guidewire for renal artery catheterization, angioplasty, and stenting under MR-guidance using MR-visible markers, in vitro and in vivo. MATERIAL AND METHODS: The new 0.035'' guidewire with fiber-reinforced PEEK core, a soft tip, and a hydrophilic coating was used. Paramagnetic markings were coated on the wire and nonbraided catheters for passive visualization. Bending stiffness of the guidewire was compared with available hydrophilic guidewires (Terumo Glidewire Stiff and Standard). A human aortic silicon phantom and 2 pigs were used. The study was animal care and use approved by the committee. Under MR-guidance, renal arteries were catheterized, balloon angioplasty was performed, and balloon expandable renal artery stents were deployed in vivo. Post mortem autopsy was performed. Guidewire visibility, pushability, steerability, and device-support capabilities of the marked guidewire were qualitatively assessed. Procedure times were recorded. RESULTS: Bending stiffness of the new PEEK-based wire was comparable with Standard Glidewire. In vitro and in vivo guidewire guidance, catheter configuration, renal artery catheterization, and balloon angioplasty were successful. In pigs, stent deployments were successful in both renal arteries. Autopsy revealed acceptable stent positioning. Guidewire visibility through applied markers was acceptable. Steerability, pushability, and device support were good in vitro and in vivo. CONCLUSIONS: The PEEK-based guide allows percutaneous MR-guided renal artery angioplasty and stenting with sufficient visibility, good steerability, pushability, and device support.

First magnetic resonance imaging-guided aortic stenting and cava filter placement using a polyetheretherketone-based magnetic resonance imaging-compatible guidewire in swine: proof of concept.

The purpose of this study was to demonstrate feasibility of percutaneous transluminal aortic stenting and cava filter placement under magnetic resonance imaging (MRI) guidance exclusively using a polyetheretherketone (PEEK)-based MRI-compatible guidewire. Percutaneous transluminal aortic stenting and cava filter placement were performed in 3 domestic swine. Procedures were performed under MRI-guidance in an open-bore 1.5-T scanner. The applied 0.035-inch guidewire has a PEEK core reinforced by fibres, floppy tip, hydrophilic coating, and paramagnetic markings for passive visualization. Through an 11F sheath, the guidewire was advanced into the abdominal (swine 1) or thoracic aorta (swine 2), and the stents were deployed. The guidewire was advanced into the inferior vena cava (swine 3), and the cava filter was deployed. Postmortem autopsy was performed. Procedural success, guidewire visibility, pushability, and stent support were qualitatively assessed by consensus. Procedure times were documented. Guidewire guidance into the abdominal and thoracic aortas and the inferior vena cava was successful. Stent deployments were successful in the abdominal (swine 1) and thoracic (swine 2) segments of the descending aorta. Cava filter positioning and deployment was successful. Autopsy documented good stent and filter positioning. Guidewire visibility through applied markers was rated acceptable for aortic stenting and good for venous filter placement. Steerability, pushability, and device support were good. The PEEK-based guidewire allows either percutaneous MRI-guided aortic stenting in the thoracic and abdominal segments of the descending aorta and filter placement in the inferior vena cava with acceptable to good device visibility and offers good steerability, pushability, and device support.

Anesthesia for cardiovascular interventions and magnetic resonance imaging in pigs.

Large animal models are still required for many experimental purposes. The aim of the current study was to define a viable narcotic procedure for experimental cardiovascular interventions and imaging in pigs. A total of 32 domestic pigs were used. Animals received propofol, midazolam, and fentanyl as continuous intravenous infusion anesthesia for complex vascular interventions, angiographic X-ray imaging, and magnetic resonance imaging (MRI). Anesthesia was maintained for 6 to 10 h. The initial hourly doses were 2.29 mg/kg of propofol, 1.14 mg/kg of midazolam, and 0.009 mg/kg of fentanyl, with controlled ventilation. Anesthesia, interventions, imaging, periods of apnea of as long as 2 min, and transportation were well-tolerated. Stress-induced arrhythmias were not noted, and artifact-free imaging was achieved. The combination of propofol, midazolam, and fentanyl is well-suited for experimental angiographic interventional studies, experimental cardiovascular MRI, and MR-guided interventions in pigs.

Inhibition of Na+/H+-exchanger with sabiporide attenuates the downregulation and uncoupling of the myocardial beta-adrenoceptor system in failing rabbit hearts.

Chronic heart failure (HF) is characterized by left ventricular (LV) structural remodeling, impaired function, increased circulating noradrenaline (NA) levels and impaired responsiveness of the myocardial beta-adrenoceptor (betaAR)-adenylyl cyclase (AC) system. In failing hearts, inhibition of the sodium/proton-exchanger (NHE)-1 attenuates LV remodeling and improves LV function. The mechanism(s) involved in these cardioprotective effects remain(s) unclear, but might involve effects on the impaired betaAR-AC system. Therefore, we investigated whether NHE-1 inhibition with sabiporide (SABI; 30 mg kg(-1) day(-1) p.o.) might affect myocardial betaAR density and AC activity in relation to changes in LV end-diastolic diameter (LVEDD) and LV systolic fractional shortening (LVS-FS) after 3 weeks of rapid LV pacing in rabbits. After 3 weeks of rapid LV pacing LVEDD was significantly increased (Shams 17+/-0.2 mm, n=9 vs 3 wksHF 20+/-0.5 mm, n=8; P<0.05) and LVS-FS decreased (Shams 31+/-1%, n=9 vs 3 wksHF 10+/-1%, n=8; P<0.05). SABI treatment significantly improved LV function independent of whether rabbits were treated after 1 week of pacing (3 wksHF+2 wksSABI (n=7): LVEDD 18+/-1 mm; LVS-FS 16+/-4%) or before pacing (3 wksHF+3wksSABI (n=9): LVEDD 18+/-1 mm; LVS-FS 18+/-6%). After 3 weeks of rapid LV pacing, SABI treatment significantly attenuated increases in serum NA content (Shams 0.83+/-0.19, 3 wksHF 2.68+/-0.38, 3 wksHF+2 wksSABI 1.22+/-0.32, 3 wksHF+3wksSABI 1.38+/-0.33 ng ml(-1)). Moreover, betaAR density (Shams 64+/-5, 3 wksHF 38+/-3, 3 wksHF+2 wksSABI 48+/-4, 3 wksHF+3 wksSABI 55+/-3 fmol mg(-1) protein) and responsiveness (isoprenaline-stimulated AC activity. (Shams 57.6+/-4.9, 3 wksHF 36.3+/-6.0, 3 wksHF+2 wksSABI 56.9+/-6.0, 3 wksHF+3 wksSABI 54.5+/-4.8 pmol cyclic AMP mg(-1) protein(-1) min(-1)) were significantly improved in SABI-treated rabbits. From the present data we cannot address whether the improved betaAR-AC system permitted improved LV function and/or whether the improved LV function resulted in less activation of the sympathetic nervous system and by this in a reduced stimulation of the betaAR-AC system. Accordingly, additional studies are needed to fully establish the cause-and-effect relationship between NHE-1 inhibition and the restoration of the myocardial betaAR system.

Feasibility of real-time magnetic resonance-guided stent-graft placement in a swine model of descending aortic dissection.

AIMS: To evaluate the pre-clinical feasibility of real-time magnetic resonance imaging (rtMRI) to guide stent-graft placement for experimental aortic dissection (AD) and to alleviate disadvantages of ionising radiation and nephrotoxic contrast media. Endovascular stent-graft placement for thoracic aortic disease is usually performed under X-ray guidance. The feasibility of rtMRI-guided stent-graft placement is currently not known. METHODS AND RESULTS: By using a catheter-based technique, dissections of the descending thoracic aorta were successfully created in eight domestic pigs. Subsequent implantation of commercially available, nitinol-based stent-grafts was performed entirely under rtMRI guidance. By pre-interventional MRI, the mean minimal true-lumen diameter was 0.9 (0.825-0.975) cm. rtMRI permitted not only the successful and safe device navigation within the true lumen from the iliac arteries to the thoracic aorta, but also the precise positioning and deployment of the stent-graft and safe withdrawal of the delivery catheter in seven of eight pigs. This was achieved without any other complications. After the stent-graft placement, MRI demonstrated complete obliteration of the false lumen, which was confirmed at autopsy. All stent-grafts were well expanded resulting in an increase in the size of the true-lumen diameter to 2.05 (1.925-2.1) cm (P=0.066 vs. baseline). CONCLUSION: In experimental AD, rtMRI-guided endovascular stent-graft placement is feasible and safe and has the potential for mitigating radiation and contrast-related side effects. Additionally, it allows not only pre-interventional diagnosis and detailed anatomic diagnosis, but also permits immediate post-interventional, anatomical, and functional delineation of procedure success that may serve as a baseline for future comparison during follow-up.

Interventional magnetic resonance angiography with no strings attached: wireless active catheter visualization.

Active instrument visualization strategies for interventional MR angiography (MRA) require vascular instruments to be equipped with some type of radiofrequency (RF) coil or dipole RF antenna for MR signal detection. Such visualization strategies traditionally necessitate a connection to the scanner with either coaxial cable or laser fibers. In order to eliminate any wire connection, RF resonators that inductively couple their signal to MR surface coils were implemented into catheters to enable wireless active instrument visualization. Instrument background to contrast-to-noise ratio was systematically investigated as a function of the excitation flip angle. Signal coupling between the catheter RF coil and surface RF coils was evaluated qualitatively and quantitatively as a function of the catheter position and orientation with regard to the static magnetic field B0 and to the surface coils. In vivo evaluation of the instruments was performed in interventional MRA procedures on five pigs under MR guidance. Cartesian and projection reconstruction TrueFISP imaging enabled simultaneous visualization of the instruments and vascular morphology in real time. The implementation of RF resonators enabled robust visualization of the catheter curvature to the very tip. Additionally, the active visualization strategy does not require any wire connection to the scanner and thus does not hamper the interventionalist during the course of an intervention.

Inhibition of the Na+/H+ exchanger attenuates the deterioration of ventricular function during pacing-induced heart failure in rabbits.

AIMS: Inhibition of the Na+/H+-exchanger (NHE) preserves myocardial morphology and function in rat and mouse models of hypertrophy and failure. The mechanism(s) involved in such cardioprotective effects remain(s) unclear, but might involve blockade of increased protein kinase activity as observed in untreated hearts. METHODS AND RESULTS: We investigated the functional, morphological and biochemical consequences of NHE-inhibition with BIIB722 in rabbits with pacing-induced heart failure (HF). In sham rabbits treated with placebo (n = 9) or BIIB722 (30 mg/kg/day po, n = 9), LV end-diastolic diameter (LVEDD) and systolic fractional shortening (FS, %) remained unchanged. In HF rabbits (n = 9), LVEDD increased and FS decreased from 31.5 +/- 1.4 to 8.1 +/- 0.9 (p < 0.05) at 3 weeks of LV pacing (400 bpm). Apoptosis, fibrosis and myocyte cross-sectional area as well as p38MAPK phosphorylation and iNOS protein expression were significantly increased in HF compared to sham rabbits. The activity of the 90 kDa NHE-kinase was greater in HF than in sham rabbits. In HF rabbits receiving BIIB722 prior to (18.1 +/- 2.2, n = 9) or following 1 week (15.5 +/- 1.6, n = 7) of pacing, FS at 3 weeks was better preserved than in untreated HF rabbits (p < 0.05). Apoptosis, fibrosis, myocyte cross-sectional area, p38MAPK phosphorylation and iNOS protein expression were significantly reduced in HF rabbits receiving BIIB722. CONCLUSION: NHE-inhibition attenuates the functional, morphological and biochemical derangements of pacing-induced HF in rabbits.

Glucocorticoid treatment prevents progressive myocardial dysfunction resulting from experimental coronary microembolization.

BACKGROUND: The frequency and importance of microembolization in patients with acute coronary syndromes and during coronary interventions have recently been appreciated. Experimental microembolization induces immediate ischemic dysfunction, which recovers within minutes. Subsequently, progressive contractile dysfunction develops over several hours and is not associated with reduced regional myocardial blood flow (perfusion-contraction mismatch) but rather with a local inflammatory reaction. We have now studied the effect of antiinflammatory glucocorticoid treatment on this progressive contractile dysfunction. METHODS AND RESULTS: Microembolization was induced by injecting microspheres (42-microm diameter) into the left circumflex coronary artery. Anesthetized dogs were followed up for 8 hours and received placebo (n=7) or methylprednisolone 30 mg/kg IV either 30 minutes before (n=7) or 30 minutes after (n=5) microembolization. In addition, chronically instrumented dogs received either placebo (n=4) or methylprednisolone (n=4) 30 minutes after microembolization and were followed up for 1 week. In acute placebo dogs, posterior systolic wall thickening was decreased from 20.0+/-2.1% (mean+/-SEM) at baseline to 5.8+/-0.6% at 8 hours after microembolization. Methylprednisolone prevented the progressive myocardial dysfunction. Increased leukocyte infiltration in the embolized myocardium was prevented only when methylprednisolone was given before microembolization. In chronic placebo dogs, progressive dysfunction recovered from 5.0+/-0.7% at 4 to 6 hours after microembolization back to baseline (19.1+/-1.6%) within 5 days. Again, methylprednisolone prevented the progressive myocardial dysfunction. CONCLUSIONS: Methylprednisolone, even when given after microembolization, prevents progressive contractile dysfunction.

TNFalpha in ischemia/reperfusion injury and heart failure.

During myocardial ischemia, both the myocardial and serum TNFalpha concentrations are rapidly increased within the area at risk. With prolongation of ischemia and development of cardiomyocyte necrosis, the TNFalpha concentration increases also in the surrounding viable portions of the myocardium. Indeed, in the scenario of myocardial ischemia/reperfusion, treatment with TNFalpha antibodies reduced the extent of myocardial infarction in rabbits and attenuated the contractile dysfunction following microembolization in dogs. In the latter studies, the serum TNFalpha concentration remained unaltered thereby supporting the notion of a direct action of TNFalpha at the level of cardiomyocytes during ischemia/reperfusion. In heart failure, the serum TNFalpha concentration is also increased, and in patients with advanced heart failure the serum TNFalpha concentration is an independent predictor of mortality. The origin of the increased serum TNFalpha concentration is not clearly identified yet, but TNFalpha derived from the heart and peripheral organs contributes to the increased serum TNFalpha concentration. Treatment with TNFalpha antibodies in the clinical scenario, however, did not improve the prognosis of heart failure patients.

Interventional MR angiography with a floating table.

A floating table was integrated into a setup for performance of interventional magnetic resonance (MR) angiography procedures with actively visualized catheters and biplanar real-time image fusion. The setup was evaluated by performing catheterizations in eight pigs. The floating table enabled the authors to follow actively visualized instruments in the pigs' vasculature during MR imaging-guided interventional angiography procedures while performing real-time biplanar MR imaging. Interventional MR angiography with a floating table enables the field of view to be moved along with the instrument tip to the region of interest and thus enhances the usability and flexibility of the interventional MR imaging setup.

Stress kinase phosphorylation is increased in pacing-induced heart failure in rabbits.

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.

Serum but not myocardial TNF-alpha concentration is increased in pacing-induced heart failure in rabbits.

In animals and patients with severe heart failure (HF), the serum tumor necrosis factor-alpha (TNF-alpha) concentration is increased. It is, however, still controversial whether or not such increased serum TNF-alpha originates from the heart itself or is of peripheral origin secondary to gastrointestinal congestion and increased endotoxin concentration. We therefore now examined TNF-alpha in serum, myocardium, and liver of sham-operated and HF rabbits. In nine rabbits in which HF was induced by left ventricular (LV) pacing at 400 beats/min for 3 wk, LV end-diastolic diameter was increased and systolic shortening fraction (9.4 +/- 1.0 vs. 28.5 +/- 1.3%, echocardiography, P < 0.05) was reduced. Serum TNF-alpha was higher in HF than in sham-operated rabbits (240 +/- 24 vs. 150 +/- 22 U/ml, WEHI-cell assay, P < 0.05). In the heart, TNF-alpha was located mainly in the vascular endothelium (immunohistochemistry), and TNF-alpha protein (920 +/- 160 vs. 900 +/- 95 U/g) did not differ between groups. In the liver of HF rabbits, hepatocytes expressed TNF-alpha, and TNF-alpha protein was increased compared with sham-operated rabbits (2,390 +/- 310 vs. 1,220 +/- 135 U/g, P < 0.05) and correlated to the number of hepatic leukocytes (r = 0.85) and serum TNF-alpha (r = 0.69). The intestinal endotoxin concentration was 24.5 +/- 1.2 vs. 17.0 +/- 3.1 endotoxin units/g wet wt (P < 0.05) in HF compared with sham-operated rabbits. In this HF model, serum but not myocardial TNF-alpha is increased. The increased serum TNF-alpha originates from peripheral sources.

Interventional MRA using actively visualized catheters, TrueFISP, and real-time image fusion.

An integrated system for performing interventional magnetic resonance angiography (MRA) with actively visualized instruments and real-time image fusion was implemented on a 1.5 T scanner. True fast imaging with steady precession (TrueFISP) imaging provided high acquisition speed paired with high signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) for the simultaneous visualization of active instruments and arterial morphology. The system enabled simultaneous image reconstruction and image postprocessing of multiple receiver channels, with subsequent image fusion display in real time. Optional interleaved image acquisition in two planes provided additional important information for biplanar instrument guidance. Various vascular interventions, including selective catheterization and subsequent selective MRA of the abdominal aorta, renal arteries, superior mesenteric artery (SMA), hepatic artery, and aortic arch, were performed on 10 pigs under MR guidance. In terms of instrument contrast, image acquisition, reconstruction, and fusion speed, the setup represents a powerful platform for performing interventional MRA procedures.