Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Thrombolyse intracoronarienne comme traitement de l'infarctus en constitution [Intracoronary thrombolysis as a treatment for evolving myocardial infarction].

Myocardial infarction is almost always the consequence of a thrombotic obstruction of one or more coronary arteries. We report our experience with the first 24 cases of intracoronary thrombolysis for recanalization of obstructed coronary arteries. 19 cases were successful, 1 case was partially successful and in 4 instances no reopening was observed. The amount of streptokinase used was 206 000 +/- 107 000 units, and reperfusion was achieved after 37 +/- 27.5 minutes. Recanalization of the vessel was accompanied by cessation of precordial pain and partial or complete normalization of the electrocardiogram. In one case bypass surgery was necessary because of reocclusion. Left ventricular function improvement after thrombolysis was dependent on the time-lag between occlusion and recanalization. These observations confirm others' experience that intracoronary thrombolysis appears to have favorable effects in patients with evolving myocardial infarction.

Carotid-subclavian arteries Index (CSI), a new echocardiographic index to recognize coarctation of the aorta in neonates and infants

Introduction: Coarctation of the aorta is a common congenital heart malformation. Mode of diagnosis changed from clinically to almost exclusively by echocardiogram and MRI. We claim to find a new echocardiographic index, based on simple and reliable morphologic measurements, to facilitate the diagnosis of aortic coarctation in the newborn.We reproduce the same procedure for older child to validate this new index. Material and Methods: We reviewed echocardiographic studies of 47 neonates with diagnosis of coarctation who underwent cardiac surgery between January 1997 and February 2003 and compared them with a matched control group. We measured 12 different sites of the aorta, aortic arch and the great vessels on the echocardiographic bands. In a second time we reviewed 23 infants for the same measurements and compare them with a matched control group. Results: 47 neonates with coarctation were analysed, age 11.8 _ 10 days,weight 3.0 _ 0.6 kg, body surface 0.20 _ 0.02m2. The control group was of 16 newborns aged 15.8 _ 10 days,weight 3.2 _ 0.9 kg and body surface 0.20 _ 0.04m2. A significant difference was noted in many morphologic measurement between the both groups, the most significant being the distance between the left carotid artery and the left subclavian artery (coarctation vs control: 7.3 _ 3mm vs 2.4 _ 0.8mm, p _ 0.0001). We then defined a new index, the carotid-subclavian arteries index (CSI) as the diameter of the distal tranverse aortic arch divided to the distance left carotid artery to left subclavian artery being also significaly different (coarctation vs control: 0.76 _ 0.86 vs 2.95 _ 1.24, p _ 0.0001). With the cutoff value of this index of 1.5 the sensitivity for aortic coarctation was 98% and the specificity of 92%. In an older group of infant with coarctation (16 patients) we apply the same principle and find for a cut-off value of 1.5 a sensitivity of 95% and a specificity of 100%. Conclusions: The CSI allows to evaluate newborns and infants for aortic coarctation with simple morphologic measurement that are not depending of the left ventricular function, presence of a patent ductus arteriosus or not. Further aggressive evaluation of these patient with a CSI _ 1.5 is indicated.

Alterations in the local myocardial motion pattern in patients suffering from pressure overload due to aortic stenosis.

BACKGROUND: MR tissue tagging allows the noninvasive assessment of the locally and temporally resolved motion pattern of the left ventricle. Alterations in cardiac torsion and diastolic relaxation of the left ventricle were studied in patients with aortic stenosis and were compared with those of healthy control subjects and championship rowers with physiological volume-overload hypertrophy. METHODS AND RESULTS: Twelve aortic stenosis patients, 11 healthy control subjects with normal left ventricular function, and 11 world-championship rowers were investigated for systolic and diastolic heart wall motion on a basal and an apical level of the myocardium. Systolic torsion and untwisting during diastole were examined by use of a novel tagging technique (CSPAMM) that provides access to systolic and diastolic motion data. In the healthy heart, the left ventricle performs a systolic wringing motion, with a counterclockwise rotation at the apex and a clockwise rotation at the base. Apical untwisting precedes diastolic filling. In the athlete's heart, torsion and untwisting remain unchanged compared with those of the control subjects. In aortic stenosis patients, torsion is significantly increased and diastolic apical untwisting is prolonged compared with those of control subjects or athletes. CONCLUSIONS: Torsional behavior as observed in pressure- and volume-overloaded hearts is consistent with current theoretical findings. A delayed diastolic untwisting in the pressure-overloaded hearts of the patients may contribute to a tendency toward diastolic dysfunction.

Thérapie cellulaire en cardiologie: bilan des premiers essais cliniques randomisés [Cell therapies in cardiology: results from the first randomized clinical trials]

Following acute myocardial infarction, necrotic cardiac tissue is replaced by scar leading to ventricular remodeling and pump failure. Transplantation of autologous bone marrow-derived cells into the heart, early post-infarct, aims to prevent ventricular remodeling. This strategy has been evaluated in four controlled, randomized clinical trials, which provided mixed results. A transient improvement in ventricular function was observed in one trial, and a modest improvement (the duration of which remains to be determined) in an additional trial, whereas two trials showed negative results. A modest benefit of bone marrow cell transplantation was also observed in patients with chronic ischemic heart disease. Despite mixed results reported so far, cell therapy of heart disease still is in its infancy and has considerable room for improvement.

Serum resistin concentrations and risk of new onset heart failure in older persons: the health, aging, and body composition (Health ABC) study.

OBJECTIVE: Resistin is associated with inflammation and insulin resistance and exerts direct effects on myocardial cells including hypertrophy and altered contraction. We investigated the association of serum resistin concentrations with risk for incident heart failure (HF) in humans. METHODS AND RESULTS: We studied 2902 older persons without prevalent HF (age, 73.6+/-2.9 years; 48.1% men; 58.8% white) enrolled in the Health, Aging, and Body Composition (Health ABC) Study. Correlation between baseline serum resistin concentrations (20.3+/-10.0 ng/mL) and clinical variables, biochemistry panel, markers of inflammation and insulin resistance, adipocytokines, and measures of adiposity was weak (all rho <0.25). During a median follow-up of 9.4 years, 341 participants (11.8%) developed HF. Resistin was strongly associated with risk for incident HF in Cox proportional hazards models controlling for clinical variables, biomarkers, and measures of adiposity (HR, 1.15 per 10.0 ng/mL in adjusted model; 95% CI, 1.05 to 1.27; P=0.003). Results were comparable across sex, race, diabetes mellitus, and prevalent and incident coronary heart disease subgroups. In participants with available left ventricular ejection fraction at HF diagnosis (265 of 341; 77.7%), association of resistin with HF risk was comparable for cases with reduced versus preserved ejection fraction. CONCLUSIONS: Serum resistin concentrations are independently associated with risk for incident HF in older persons.

Cardiac rotation and relaxation in patients with aortic valve stenosis.

BACKGROUND: Diastolic dysfunction with delayed relaxation and abnormal passive elastic properties has been described in patients with severe pressure overload hypertrophy. The purpose of this study was to evaluate the time course of rotational motion of the left ventricle in patients with aortic valve stenosis using myocardial tagging. METHODS: Myocardial tagging is a non-invasive method based on magnetic resonance which makes it possible to label ('tag') specific myocardial regions. From the motion of the tag's cardiac rotation, radial displacement and translational motion can be determined. In 12 controls and 13 patients with severe aortic valve stenosis systolic and diastolic wall motion was assessed in an apical and basal short axis plane. RESULTS: The normal left ventricle performs a systolic wringing motion around the ventricular long axis with clockwise rotation at the base (-4.4+/-1.6 degrees) and counter-clockwise rotation at the apex (+6.8+/-2.5 degrees) when viewed from the apex. During early diastole an untwisting motion can be observed which precedes diastolic filling. In patients with aortic valve stenosis systolic rotation is reduced at the base (-2.4+/-2.0 degrees; P<0.01) but increased at the apex (+12.0+/-6.0 degrees; P<0.05). Diastolic untwisting is delayed and prolonged with a decrease in normalized rotation velocity (-6.9+/-1.1 s(-1)) when compared to controls (-10.7+/-2.2 s(-1); P<0.001). Maximal systolic torsion is 8.0+/-2.1 degrees in controls and 14.1+/-6.4 degrees (P<0.01) in patients with aortic valve stenosis. CONCLUSIONS: Left ventricular pressure overload hypertrophy is associated with a reduction in basal and an increase in apical rotation resulting in increased torsion of the ventricle. Diastolic untwisting is delayed and prolonged. This may explain the occurrence of diastolic dysfunction in patients with severe pressure overload hypertrophy.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Rivascolarizzazione miocardica con arteria radiale: tecniche chirurgiche a confronto e risultati a breve termine [Myocardial revascularization and radial artery: different surgical strategies and short-term results]

BACKGROUND: The radial artery is routinely used as a graft for surgical arterial myocardial revascularization. The proximal radial artery anastomosis site remains unknown. In this study, we analyzed the short-term results and the operative risk determinants after having used four different common techniques for radial artery implantation. METHODS: From January 2000 to December 2004, 571 patients underwent coronary artery bypass grafting with radial arteries. Data were analyzed for the entire population and for subgroups following the proximal radial artery anastomosis site: 140 T-graft with the mammary artery (group A), 316 free-grafts with the proximal anastomosis to the ascending aorta (group B), 55 mammary arteries in situ elongated with the radial artery (group C) and 60 radial arteries elongated with a piece of mammary artery and anastomosed to the ascending aorta (group D). RESULTS: The mean age was 53.8 +/- 7.7 years; 55.5% of patients had a previous myocardial infarction and 73% presented with a satisfactory left ventricular function. A complete arterial myocardial revascularization was achieved in 532 cases (93.2%) and 90.2% of the procedures were performed under cardiopulmonary bypass and cardioplegic arrest. The operative mortality rate was 0.9%, a postoperative myocardial infarction was diagnosed in 19 patients (3.3%), an intra-aortic balloon pump was used in 10 patients (1.7%) and a mechanical circulatory device was implanted in 2 patients. The radial artery harvesting site remained always free from complications. The proximal radial artery anastomosis site was not a determinant of early hospital mortality. Group C showed a higher risk of postoperative myocardial infarction (p = 0.09), together with female gender (p = 0.003), hypertension (p = 0.059) and a longer cardiopulmonary bypass time. CONCLUSIONS: The radial artery and the mammary artery can guarantee multiple arterial revascularization also for patients with contraindications to double mammary artery use. The four most common techniques for proximal radial artery anastomosis are not related to a higher operative risk and they can be used alternatively to reach the best surgical results

Connexin 43 expression in human hypertrophied heart due to pressure and volume overload.

Left ventricular hypertrophy (LVH) is due to pressure overload or mechanical stretch and is thought to be associated with remodeling of gap-junctions. We investigated whether the expression of connexin 43 (Cx43) is altered in humans in response to different degrees of LVH. The expression of Cx43 was analyzed by quantitative polymerase chain reaction, Western blot analysis and immunohistochemistry on left ventricular biopsies from patients undergoing aortic or mitral valve replacement. Three groups were analyzed: patients with aortic stenosis with severe LVH (n=9) versus only mild LVH (n=7), and patients with LVH caused by mitral regurgitation (n=5). Cx43 mRNA expression and protein expression were similar in the three groups studied. Furthermore, immunohistochemistry revealed no change in Cx43 distribution. We can conclude that when compared with mild LVH or with LVH due to volume overload, severe LVH due to chronic pressure overload is not accompanied by detectable changes of Cx43 expression or spatial distribution.

Troubleshooting in extracorporeal life support.

Since the first clinical use of extracorporeal circulation in the last century [1] by John Gibbon and the first successful mechanical support of the left ventricular function by Forest Dodrill [2], the progress of techniques and technologies has helped to develop minimised systems for extracorporeal circulatory and respiratory support. However, the fact is that, despite the advanced technologies used for extracorporeal support, successful application in order to be benefit a critically ill population requires highly trained and skilled teams. Application of these highly sophisticated techniques in life-saving situations inside and/or outside the operating room is a procedure with certain pitfalls and dangers. The aim of this review is to provide a short overview of the technical aspects of extracorporeal circulation, with a look at the recent literature and clinical experiences focusing on technical as well surgical considerations regarding the urgent and/or emergent usage of a central as well as peripheral extracorporeal system.

Lentiviral vectors efficiently transduce the EGFP gene into cardiomyocytes in vivo : immunohistology and Elisa quantification of the transgene

RESUME Les maladies cardio-vasculaires représentent la cause la plus importante de mortalité et de morbidité dans les pays occidentaux. La thérapie génique offre une nouvelle approche au traitement de ces maladies. L'expression de gènes protecteurs dans le myocarde par des technologies de transfert génique peut améliorer la fonction ventriculaire lors de l'insuffisance cardiaque ou stimuler la formation de nouveaux vaisseaux dans la maladie coronarienne. Etant donné qu'une majorité des maladies cardiaques sont des maladies chroniques, l'expression durable du gène thérapeutique introduit dans le coeur est souhaitable dans de nombreux cas. Malheureusement, l'utilité des vecteurs de transfert génique les plus utilisés en thérapie génique cardiovasculaire est limitée par une performance faible (ADN plasmidique) et une courte durée d'expression (adénovirus). Récemment, des vecteurs de transfert génique dérivés des lentivirus, une sous-famille des rétrovirus, ont retenu l'attention de la communauté scientifique en raison de leur capacité à exprimer des gènes à long terme. Contrairement aux vecteurs rétroviraux traditionnels, les vecteurs lentiviraux transduisent des gènes même dans des cellules qui ne se divisent pas, ce qui est le cas des cardiomyocytes adultes. Ces vecteurs présentent un profil de biosécurité comparable à celui des vecteurs rétroviraux traditionnels. Nous avons donc décidé de tester l'utilité des vecteurs lentiviraux pour le transfert génique dans des cardiomyocytes de rat adulte in vitro et in vivo. Plusieurs versions de vecteurs lentiviraux contenant différent promoteurs ont été construites. Ces vecteurs contenant le gène marqueur EGFP (enhanced green fluorescent protein) ont été testés dans des cardiomyocytes de rat in vitro, ainsi que dans des coeurs de rat in vivo. Le but de ces expériences était de déterminer la durée de l'expression du transgène après injection intramyocardique chez le rat. Pour ce faire, nous avons développé une technique ELISA pour détecter la protéine EGFP dans des extraits de tissu cardiaque. Les résultats ont montré que la protéine EGFP était encore présente à des niveaux significatifs jusqu'à dix semaines après l'injection de vecteurs lentiviraux, alors que l'expression transgénique obtenue avec un vecteur adénoviral traditionnel a été plus limitée dans le temps. Ces résultats démontrent la capacité des vecteurs lentiviraux à exprimer des gènes d'intérêt de manière performante et stable dans le cœur de rat adulte in vivo. SUMMARY Cardiovascular diseases are the first cause of morbidity and mortality in Western countries. Gene therapy offers a new approach to these diseases. Expression of therapeutic genes in the myocardium by gene transfer technologies can improve ventricular function in heart failure and stimulate neovascularization in coronary disease. Chronic heart diseases likely require sustained expression of the therapeutic gene within the heart itself. Unfortunately, the most commonly used vectors in cardiovascular gene therapy, i.e. plasmid DNA and recombinant adenovirus vectors, are limited by poor DNA uptake and transient transgene expression, respectively. Recently, lentivirus-derived vectors have attracted much interest because of their ability to achieve long-term transgene expression. In contrast to traditional retroviral vectors, lentiviral vectors are also able to transduce non- dividing cells, while presenting a comparable biosafety profile. Adult cardiomyocytes are terminally differentiated cells that do not divide under normal conditions. For these reasons, we have decided to evaluate the efficiency of lentiviral vectors for gene-transduction of adult cardiomyocytes both in vitro and in vivo. We constructed various types of lentiviral vectors containing various promoters. Vectors encoding EGFP as a reporter gene were tested in rat cardiomyocytes in vitro and in rat hearts in vivo. The aim of the experiments involved in this thesis work was to determine the duration of the expression of the transgene after rat intramyocardial injection using a quantitative assay. Therefore, an ELISA technique was set up to measure the EGFP protein in rat heart tissue extracts. Our results showed that the EGFP protein was still present at significant levels at ten weeks after lentiviral vector injection, whereas the duration of expression with adenoviral vectors was shorter. These results demonstrate that lentiviral vectors efficiently deliver genes and achieve sustained transgene expression in adult rat cardiomyocytes in vivo.

Obstructive pulmonary artery sarcoma.

A 68 year-old woman presented with increasing dyspnoea (NYHA II) and systolic murmur at auscultation. Echocardiography showed thickened pulmonary valve leaflets, a systolic prolapsing mass provoking severe pulmonary stenosis (peak systolic pulmonary pressure: 42 mmHg), no regurgitation, minimal right ventricular dilatation but normal ventricular function. CT scan showed a dense structure extending from the right ventricular outflow tract (RVOT) up to the pulmonary bifurcation infiltrating the pulmonary valve (PV).

Free-running 4D whole-heart self-navigated golden angle MRI: Initial results.

PURPOSE: To test the hypothesis that both coronary anatomy and ventricular function can be assessed simultaneously using a single four-dimensional (4D) acquisition. METHODS: A free-running 4D whole-heart self-navigated acquisition incorporating a golden angle radial trajectory was implemented and tested in vivo in nine healthy adult human subjects. Coronary magnetic resonance angiography (MRA) datasets with retrospective selection of acquisition window width and position were extracted and quantitatively compared with baseline self-navigated electrocardiography (ECG) -triggered coronary MRA. From the 4D datasets, the left-ventricular end-systolic, end-diastolic volumes (ESV & EDV) and ejection fraction (EF) were computed and compared with values obtained from conventional 2D cine images. RESULTS: The 4D datasets enabled dynamic assessment of the whole heart with isotropic spatial resolution of 1.15 mm(3) . Coronary artery image quality was very similar to that of the ECG-triggered baseline scan despite some SNR penalty. A good agreement between 4D and 2D cine imaging was found for EDV, ESV, and EF. CONCLUSION: The hypothesis that both coronary anatomy and ventricular function can be assessed simultaneously in vivo has been tested positive. Retrospective and flexible acquisition window selection allows to best visualize each coronary segment at its individual time point of quiescence. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.