Fanny Vaillant

Heart lesions associated with anabolic steroid abuse: Comparison of post-mortem findings in athletes and norethandrolone-induced lesions in rabbits

Among 15,000 forensic post-mortem examinations performed on the coroners order over a 24-year period (January 1981-December 2004) in the area of Lyon, France (population: 2,000,000), 2250 cases of unexpected cardiac sudden death were identified retrospectively according to WHO criteria. Of these, 108 occurred during recreational sport and 12 occurred in athletes. In the latter category, a history of anabolic steroid abuse was found in 6 cases, whereas pre-existing ordinary cardiac lesions were observed in the 6 remaining cases. To shed light on the possible role of anabolic steroids in the induction of cardiac lesions, an experimental study was conducted in rabbits that were treated orally with norethandrolone 8mg/kg/day for 60 days, and sacrificed at day 90. The histopathological examination of the heart from treated animals showed coronary thrombosis associated with left ventricle hypertrophy in 3 cases, and lesions analogous to toxic or adrenergic myocarditis in all other treated animals. These findings were very similar to those observed after cardiac sudden death in the 6 athletes with a history of anabolic steroid abuse. In addition, elevated caspase-3 activity in the heart of treated rabbits as compared to controls suggests that apoptosis is involved in the induction of norethandrolone-induced cardiac lesions. These results confirm the cardiotoxic potential of anabolic steroid abuse.

Heart rate reduction with ivabradine increases ischaemia-induced ventricular fibrillation threshold: Role of myocyte structure and myocardial perfusion☆

AIMS We showed previously that ivabradine (IVA), a selective inhibitor of the cardiac pacemaker I(f) current, achieved protection against ischaemia-induced ventricular fibrillation (VF) in pigs by increasing the VF threshold (VFT). This was correlated to the heart rate reduction (HRR), the limitation of monophasic action potential shortening and the reduction of the hypoxic area. This study investigated myocyte ultrastructure and regional myocardial blood flow (RMBF), potentially involved in these cardioprotective effects of IVA. METHODS AND RESULTS Myocardial ischaemia was induced in pigs by total 1-min occlusion of the left anterior descending coronary artery following i.v. administration of saline (n=6) or IVA (0.25 mg/kg, n=6). Electrophysiological and haemodynamic parameters, the hypoxic area and the presence of myocyte ultrastructural lesions were evaluated. The RMBF was assessed using positron emission tomography following ischaemia/reperfusion in IVA (0.25 mg/kg, i.v., n=6) or vagal stimulation (n=4) groups. Compared with saline, IVA induced a 32% HRR (p<0.01), a 2.9-fold increase in the VFT (p<0.001) and a reduction of the hypoxic area without any change in left ventricular dP/dt(max). IVA preserved cardiomyocyte morphology, particularly mitochondrial ultrastructure. Compared with baseline, RMBF during reperfusion was increased in the hypoxic area following IVA administration (+218% vs. +97%, p<0.05) or vagal stimulation (+195% vs. +127%, p<0.05). This increase was sharply reduced by atrial pacing in IVA-group. CONCLUSION IVA exerts a cardioprotection from ischaemia-induced VF by increasing RMBF and preserving cardiomyocyte and mitochondrial ultrastructure, which opens new perspectives regarding potential targets that would be involved in the anti-ischaemic effects of IVA.

Protective role of selenium supplementation against cardiac lesions induced by the combination of levomepromazine and risperidone in the rabbit

Neuroleptics are a suspected cause of sudden death in psychiatric patients, especially in those with pre-existing cardiac lesions. As these lesions were previously shown to be associated with selenium (Se) deficiency, the aim of the present study was to evidence the possible protective effect of Se supplementation against cardiac lesions induced by the combination of the neuroleptic drugs levomepromazine and risperidone in the rabbit. Two groups of 6 rabbits were treated with 3 mg/kg of levomepromazine daily intramuscularly combined with 1 mg/kg of risperidone intramuscularly every other week for 3 consecutive months, and one group additionally received a solution of sodium selenite (2 μg/kg/day) intramuscularly during the whole treatment period. Furthermore, one group of six untreated animals was given the Se supplementation and another group of six control animals received saline daily. Blood samples were drawn before and at the end of the treatment period for the measurement of serum Se levels. At the end of the study, all animals were sacrificed and their hearts were removed for the measurement of tissue Se concentrations. In addition, the hearts were prepared for histopathological examination. A variety of cardiac lesions was found in the neuroleptics-treated animals without supplementation and to a lesser extent in the control and Se-supplemented untreated animals. Importantly, only rare cardiac lesions were observed in neuroleptics-Se-treated animals. The most striking differences in Se concentrations were noted in the myocardium: as compared to controls, there was a 43% reduction in neuroleptics-treated, but non-Se-supplemented animals (p < .01), at the end of the treatment period, whereas only a 14% reduction (p < .05) was noted in the neuroleptics-Se-treated animals. These results confirm that neuroleptics induce cardiac lesions associated with Se deficiency. Selenium supplementation markedly decreased the incidence and severity of neuroleptics-induced cardiac lesions and these findings may serve as a basis for further evaluation of the protective role of Se supplementation in neuroleptics-treated patients. However, Se supplementation in normal animals without Se deficiency was also shown to be cardiotoxic.

Non-invasive cardiac pacing with image-guided focused ultrasound

Currently, no non-invasive cardiac pacing device acceptable for prolonged use in conscious patients exists. High Intensity Focused Ultrasound (HIFU) can be used to perform remote pacing using reversibility of electromechanical coupling of cardiomyocytes. Here we described an extracorporeal cardiac stimulation device and study its efficacy and safety. We conducted experiments ex vivo and in vivo in a large animal model (pig) to evaluate clinical potential of such a technique. The stimulation threshold was determined in 10 different ex vivo hearts and different clinically relevant electrical effects such as consecutive stimulations of different heart chambers with a single ultrasonic probe, continuous pacing or the inducibility of ventricular tachycardia were shown. Using ultrasonic contrast agent, consistent cardiac stimulation was achievable in vivo for up to 1 hour sessions in 4 different animals. No damage was observed in inversion-recovery MR sequences performed in vivo in the 4 animals. Histological analysis revealed no differences between stimulated and control regions, for all ex vivo and in vivo cases.

0514 : Isolation of cardiac myocytes from human heart

Background The investigation of single cardiac myocytes from healthy and diseased hearts of various species is a valuable tool to explore cardiac physio/ pathophysiology. The application of cell isolation to human donor tissue has been proofed to be difficult due to the limited amount of human tissue (mainly human right atrial appendages during cardiac surgery). Another limitation is the low viability of cardiomyocytes after isolation. In this study, we present a method to obtain single cardiac myocytes from different regions of human heart. Methods and results Human hearts rejected for transplantation were obtained from Bordeaux hospital. This protocol was approved by the Agence de la Biomedecine. Left atrial (LA) and ventricular (LV) myocytes were obtained by enzymatic dissociation. The ventricles and right atrium were removed and used for other studies (e.g. high resolution optical mapping). LA was cannulated by the circumflex artery and mounted into a Langendorff perfusion system after suture of the leaky atrial branches. LA was perfused with a Ca2+-free solution (~10 min), then collagenase and protease solution (0.08 mM Ca2+) and recirculated for ~25 min. Enzymes were washed out with a 0.2 mM Ca2+ solution. LA was separated into 4 regions: Endocardium, Epicardium, roof and pulmonary vein; LV myocytes were also obtained. Cells were re-suspended into a 1.8 mM Ca2+ solution by steps. Ca2+ transients were recorded (Fura-2, field stimulation) using an IonOptix system and cell membrane was stained with di-8 ANEPPS and visualized under confocal microscopy. Ca2+ tolerant myocytes were obtained from the 4 LA regions and LV. Human cardiac myocytes respond to electrical stimulation and Ca2+ transient can be recorded. Analysis of functional and structural data will be presented. Conclusion Isolation of single cardiac myocytes from human samples is a tedious task, but we present data showing reliable method to obtain functional and structural insights. The author hereby declares no conflict of interest

0291 : Mitochondrial permeability transition pore opening might contribute to ventricular arrhythmia maintenance

Background Calcium leak from the sarcoplasmic reticulum (SR) favors cardiac sudden death through a calcium overload mechanism. Here we investigate the crosstalk between the calcium overload SR-mediated and the mitochondrial permeability transition pore (mPTP) opening during a ventricular arrhythmic episode. Methods and results a burst pacing protocol was developed to induce ventricular arrhythmia in the perfused rat heart. Surface electrocardiogram, left ventricular pressure, and oxygen consumption were monitored. Epicardial fluorescence was recorded from either a mitochondrial membrane potential dye or an intracellular calcium dye. ECG signals were used to assess both arrhythmia complexity and duration. Hearts were perfused with tacrolimus (FK506) alone or in combination with cyclosporin A (CsA) to promote SR calcium leak or inhibit mPTP, respectively. Prior to arrhythmia induction, FK506 promoted mitochondrial depolarization and intracellular calcium overload in a CsA-sensitive manner. More than 90% of hearts developed complex ventricular arrhythmias in the FK506 group and this was accompanied by a strong mitochondrial membrane depolarization. In contrast, in the FK506 + CsA group, 100% of the hearts spontaneously defibrillate after a few seconds of arrhythmias. In this group, following arrhythmia induction, mitochondrial depolarization amplitude, intracellular calcium overload and heart efficiency depression was decreased in comparison to the FK506 group. Conclusions Here we suggest that the FK506-induced calcium overload promotes mitochondrial calcium accumulation and leads to mPTP opening. We propose that mPTP opening should be considered as an arrhythmogenic substrate. The author hereby declares no conflict of interest

0176: In vitro study of nucleotide adenylic compartmentation in the four cardiac chambers

Balance between energy supply and demand is finely tuned in heart. In beating heart, intrinsic homeostasis depends on the parallel activation of both modules. Here we used the in vitro approach recently introduced in the Institute (the permeabilized skinned fiber) to study energetics of the four different cardiac chambers (right and left atria, right and left ventricles) in order to evidence local differences in the energetics regulation. Using high-resolution oxygraphy, mitochondrial function was quantified and apparent affinities of mitochondrial respiration for added ADP and ATP were obtained in absence or not of 25μM-blebbistatin (myosin ATPase inhibitor). Measurement of ATP hydrolysis by spectrofluorometry was also experimented in presence or not of blebbistatin after inhibition of mitochondrial oxidative phosphorylation (oligomycin, ATP synthase inhibitor) and respiratory chain inhibitor (rotenone). Apparent affinities of mitochondrial oxidative phosphorylation for ADP were significantly higher in ventricles compared to atria. Presence of blebbistatin induced a significant increase in the affinity in left atrium, underlining the small mitochondrial sensitivity to ADP in the case of myosin ATPase inhibition. Blebbistatin addition under condition of mitochondrial inhibition induced a 50% inhibition of ATP hydrolysis in the left ventricle defining an interesting specific regulation in this compartment. We conclude on the hypothesis of different “channeling” mechanisms between energy supply and energy demand in each cardiac compartments. Our results clearly show a weaker adaptability in energy regulation in the left atrium compared to the left ventricle. A model of failing rat heart is now under study in order to explore the implication of nucleotide adenylic compartimentation on pathological electric instability.