Cardiovascular diseases represent an economic burden for health systems accounting for substantial morbidity and mortality worldwide. Despite timely and costly efforts in drug development, the cardiovascular safety and efficacy of the drugs are not always fully achieved. These lead to the drugs' withdrawal with adverse cardiac effects from the market or in the late stages of drug development. There is a growing need for a cost-effective drug screening assay to rapidly detect potential acute drug cardiotoxicity. The Langendorff isolated heart perfusion technique, which provides cardiac hemodynamic parameters (e.g., contractile function and heart rate), has become a powerful approach in the early drug discovery phase to overcome drawbacks in the drug candidate's identification. However, traditional ex vivo retrograde heart perfusion methods consume a large volume of perfusate, which increases the cost and limits compound screening. An elegant and cost-effective alternative mode for ex vivo retrograde heart perfusion is the constant-flow with a recirculating circuit (CFCC), which allows assessment of cardiac function using a reduced perfusion volume while limiting adverse effects on the heart. Here, we provide evidence for cardiac parameters stability over time in this mode. Next, we demonstrate that our recycled ex vivo perfusion system and the traditional open one yield similar outputs on cardiac function under basal conditions and upon ?-adrenergic stimulation with isoproterenol. Subsequently, we validate the proof of concept of therapeutic agent screening using this efficient method. ?-blocker (i.e., propranolol) infusion in closed circulation countered the positive effects induced by isoproterenol stimulation on cardiac function. Keywords: Drug development, Drug screening, Cardiovascular safety, Langendorff method, Closed circulation.
- MeSH
- Isoproterenol pharmacology MeSH
- Rats MeSH
- Perfusion * methods MeSH
- Drug Evaluation, Preclinical methods MeSH
- Isolated Heart Preparation * methods MeSH
- Heart * drug effects physiology MeSH
- Heart Rate drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Isoproterenol MeSH
1. Definition. The isolated perfused heart according to Langendorff is a preparation which is easily obtained from all warm-blooded animals. The heart is usually perfused at constant pressure, but frequently at constant flow, mostly with oxygenated saline solutions. The ventricles do not fill with the perfusate and therefore do not perform pressure-volume work. If, however, an intraventricular balloon is inserted, the ventricle can contract isovolumetrically. 2. Measuring parameters. Mechanical parameters of the working myocardium (contractile force, volume, ventricular diameter), mean coronary flow, bioelectrical parameters (EKG, monophasic injury potentials), and cardiac rhythm can be measured and recorded. 3. Function tests. The intact function of the working myocardium and the coronary vessels should be examined using various simple tests regarding the myocardium, the smooth musculature, and the endothelium of coronary vessels. 4. Possible applications. In the Langendorff-heart of normal or pretreated animals, inotropic, chronotropic, antiarrhythmic or vasoactive substances can be investigated in the steady state or by means of specific stress tests. In addition, the preparation is particularly suitable for biochemical studies of myocardial metabolism. 6. Advantages. Due to the technical ease with which a variety of parameters can be measured, a single heart can provide maximum information about the condition of the myocardium and the coronary vessels. Thus, it is not surprising to find that 90 years after its first description, the isolated perfused heart according to Langendorff continues to be utilized.
- MeSH
- Electrophysiology MeSH
- Heart Function Tests MeSH
- Myocardial Contraction MeSH
- Coronary Circulation MeSH
- Perfusion methods MeSH
- Mammals MeSH
- Heart physiology MeSH
- Heart Rate MeSH
- In Vitro Techniques MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.
- MeSH
- RNA, Messenger genetics MeSH
- MicroRNAs metabolism MeSH
- Mice MeSH
- Polyribosomes metabolism MeSH
- Proteomics methods MeSH
- Heart growth & development MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Video-Audio Media MeSH
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Names of Substances
- RNA, Messenger MeSH
- MicroRNAs MeSH
Atrioventricular (AV) accessory pathways (APs) provide additional electrical connections between the atria and ventricles, resulting in severe electrical disturbances. It is generally accepted that APs originate in the altered annulus fibrosus maturation in the late prenatal and perinatal period. However, current experimental methods cannot address their development in specific locations around the annulus fibrosus because of the inaccessibility of late fetal hearts for electrophysiological investigation under physiological conditions. In this study, we describe an approach for optical mapping of the retrogradely perfused chick heart in the last third of the incubation period. This system showed stability for electrophysiological measurement for several hours. This feature allowed analysis of the number and functionality of the APs separately in each clinically relevant position. Under physiological conditions, we also recorded the shortening of the AV delay with annulus fibrosus maturation and analyzed ventricular activation patterns after conduction through APs at specific locations. We observed a gradual regression of AP with an area-specific rate (left-sided APs disappeared first). The results also revealed a sudden drop in the number of active APs between embryonic days 16 and 18. Accessory myocardial AV connections were histologically documented in all positions around the annulus fibrosus even after hatching. The fact that no electrically active AP was present at this stage highlights the necessity of electrophysiological evaluation of accessory atrioventricular connections in studying AP formation.NEW & NOTEWORTHY We present the use of retrograde perfusion and optical mapping to investigate, for the first time, the regression of accessory pathways during annulus fibrosus maturation, separately examining each clinically relevant location. The system enables measurements under physiological conditions and demonstrates long-lasting stability compared with other approaches. This study offers applications of the model to investigate electrical and/or functional development in late embryonic development without concern about heart viability.
- Keywords
- Langendorff perfusion, atrioventricular accessory pathway, preexcitation,
- MeSH
- Action Potentials * MeSH
- Chick Embryo MeSH
- Atrioventricular Node embryology physiopathology MeSH
- Perfusion MeSH
- Animals MeSH
- Check Tag
- Chick Embryo MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
OBJECTIVES: To establish the optimal machine perfusion temperature for recovery of hearts in a rodent model of donation after declaration of cardiocirculatory death (DCD). METHODS: Hearts from male Lewis rats (n = 14/group) were subjected to 25 min of in situ warm (37°C) ischaemia to simulate DCD. They were then explanted and reperfused with diluted autologous blood for 60 min at 20, 25, 30, 33 or 37°C, after which they were stored at 0-4°C in Custodiol preservation solution for 240 min. Fresh-excised and cold-stored ischaemic hearts were used as controls. The viability of the different groups was assessed by comparing heart rate and left ventricular contractility in a Langendorff circuit, as well as perfusate levels of troponin-t and creatine kinase (CK), and myocardial levels of adenosine triphosphate (ATP) and reduced glutathione. RESULTS: During ex vivo reperfusion, hearts in all groups resumed beating within minutes. The mean heart rate was highest in the 37°C group at 154.72 ± 33.01 beats × min(-1) (bpm), and declined in proportion to temperature to 39.72 ± 5.53 bpm at 20°C. Troponin-t levels were highest in the 37°C group (79.49 ± 20.79 µg/l), the values were significantly lower in all other reconditioned groups with a minimum of 12.472 ± 7.08 µg/l in the 20°C group (P < 0.0001). Tissue ATP levels ranged from 4.32 ± 1.71 µmol/g at 33°C to 4.59 ± 1.41 µmol/g at 30°C, all significantly higher than the mean ATP level of 1.41 ± 0.93 µmol/g in untreated ischaemic hearts (P < 0.0001). During Langendorff assessment, the mean heart rate and contractility of all groups were higher than those of cold-stored ischaemic hearts (P < 0.0001), yet not significantly different from those of fresh controls. The perfusate levels of troponin-t and CK, and myocardial levels of reduced-glutathione and ATP were not significantly different between groups. CONCLUSION: Our results suggest that mild hypothermia during ex vivo reperfusion improves recovery of ischaemic hearts in a rodent DCD model.
- Keywords
- Declaration of cardiocirculatory death, Heart transplantation, Ischaemia-reperfusion, Machine perfusion, Organ preservation,
- MeSH
- Tissue Donors MeSH
- Rats MeSH
- Survival Rate MeSH
- Disease Models, Animal MeSH
- Random Allocation MeSH
- Recovery of Function MeSH
- Tissue and Organ Harvesting methods MeSH
- Rats, Inbred Lew MeSH
- Graft Survival MeSH
- Myocardial Reperfusion methods MeSH
- Death * MeSH
- Cold Ischemia methods MeSH
- In Vitro Techniques MeSH
- Hypothermia, Induced methods MeSH
- Heart Transplantation methods MeSH
- Organ Preservation methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
AIM: The present study was designed to investigate the effects of estradiol-ethylenediamine derivative on perfusion pressure and coronary resistance in rats. An additional aim was to identify the molecular mechanisms involved. METHODS: The Langendorff model was used to measure perfusion pressure and coronary resistance changes in isolated rat heart after estradiol-ethylenediamine derivative alone and following compounds; tamoxifen (estrogen receptor antagonist), prazosin (alpha1 adrenoreceptor antagonist), metoprolol (selective beta1 receptor blocker), indomethacin (prostanglandin synthesis inhibitor) and nifedipine (L-type calcium-channel inhibitor). RESULTS: The results show that estradiol-ethylenediamine derivative [10(-9) mmol] significantly increased perfusion pressure (p = 0.005) and coronary resistance (p = 0.006) in isolated rat heart. Additionally, the effect of estradiolethylenediamine on perfusion pressure [10(-9) to 10(-4) mmol] was only blocked in the presence of the L-type calcium-channel (nifedipine). CONCLUSIONS: These data suggest that the effect of estradiol-ethylenediamine on perfusion pressure and vascular coronary involves activation of the L-type calcium channel through a non-genomic molecular mechanism.
- MeSH
- Adrenergic alpha-1 Receptor Antagonists pharmacology MeSH
- Adrenergic beta-1 Receptor Antagonists pharmacology MeSH
- Estrogen Antagonists pharmacology MeSH
- Calcium Channel Blockers pharmacology MeSH
- Vascular Resistance drug effects MeSH
- Estradiol analogs & derivatives pharmacology MeSH
- Ethylenediamines pharmacology MeSH
- Indomethacin pharmacology MeSH
- Cyclooxygenase Inhibitors pharmacology MeSH
- Coronary Circulation drug effects MeSH
- Blood Pressure drug effects MeSH
- Rats MeSH
- Metoprolol pharmacology MeSH
- Myocardium metabolism MeSH
- Nifedipine pharmacology MeSH
- Rats, Wistar MeSH
- Prazosin pharmacology MeSH
- Tamoxifen pharmacology MeSH
- In Vitro Techniques MeSH
- Calcium Channels, L-Type metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Adrenergic alpha-1 Receptor Antagonists MeSH
- Adrenergic beta-1 Receptor Antagonists MeSH
- Estrogen Antagonists MeSH
- Calcium Channel Blockers MeSH
- Estradiol MeSH
- Ethylenediamines MeSH
- Indomethacin MeSH
- Cyclooxygenase Inhibitors MeSH
- Metoprolol MeSH
- Nifedipine MeSH
- Prazosin MeSH
- Tamoxifen MeSH
- Calcium Channels, L-Type MeSH
AIM: The present study was designed to investigate the effects of progesterone-carbachol derivative on perfusion pressure and coronary resistance in rats. An additional aim was to identify the molecular mechanisms involved. METHODS: The Langendorff model was used to measure perfusion pressure and coronary resistance changes in isolated rat heart after progesterone-carbachol derivative alone and after the following compounds; mifepristone (progesterone receptor blocker), yohimbine (α2 adreno-receptor antagonist), ICI 118,551 (selective β2 receptor blocker), atropine (non-selective muscarinic receptor antagonist), methoctramine (antagonist of M2 receptor) and L-NAME (inhibitor of nitric oxide synthase). RESULTS: The results show that progesterone-carbachol derivative [10(-9) mM] significantly decreased perfusion pressure (P=0.005) and coronary resistance (P=0.006) in isolated rat heart. Additionally, the effect of progesterone-carbachol on perfusion pressure [10(-9) to 10(-4) mM] was only blocked in the presence of methoctramine and L-NAME. CONCLUSIONS: These data suggest that progesterone derivative exert its effect on perfusion pressure via activation of the M2 muscarinic. In addition, this phenomenon involves stimulation of nitric oxide synthase (NOS).
- MeSH
- Vascular Resistance drug effects MeSH
- Carbachol analogs & derivatives pharmacology MeSH
- Coronary Vessels drug effects physiology MeSH
- Blood Pressure drug effects MeSH
- Rats MeSH
- Progesterone analogs & derivatives pharmacology MeSH
- Receptor, Muscarinic M2 drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- 17-acetyl-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydrocyclopenta(a)phenanthren-3-ylidenecarbamoyloxy)ethyl)trimethylammonium MeSH Browser
- Carbachol MeSH
- Progesterone MeSH
- Receptor, Muscarinic M2 MeSH
Acute streptozotocin diabetes mellitus (DM) as well as remote ischemic preconditioning (RPC) has shown a favorable effect on the postischemic-reperfusion function of the myocardium. Cardioprotective mechanisms offered by these experimental models involve the mitochondria with the changes in functional properties of membrane as the end-effector. The aim was to find out whether separate effects of RPC and DM would stimulate the mechanisms of cardioprotection to a maximal level or whether RPC and DM conditions would cooperate in stimulation of cardioprotection. Experiments were performed on male Wistar rats divided into groups: control, DM, RPC and DM treated by RPC (RPC+DM). RPC protocol of 3 cycles of 5-min hind limb ischemia followed by 5-min reperfusion was used. Ischemic-reperfusion injury was induced by 30-min ischemia followed by 40-min reperfusion of the hearts in Langendorff mode. Mitochondria were isolated by differential centrifugation, infarct size assessed by staining with 1 % 2,3,5-triphenyltetrazolium chloride, mitochondrial membrane fluidity with a fluorescent probe DPH, CoQ(9) and CoQ(10) with HPLC. Results revealed that RPC as well as DM decreased the infarct size and preserved mitochondrial function by increasing the mitochondrial membrane fluidity. Both used models separately offered a sufficient protection against ischemic-reperfusion injury without an additive effect of their combination.
- MeSH
- Time Factors MeSH
- Diabetes Mellitus, Experimental chemically induced metabolism pathology MeSH
- Membrane Fluidity MeSH
- Adaptation, Physiological MeSH
- Myocardial Infarction metabolism pathology prevention & control MeSH
- Mitochondrial Membranes pathology MeSH
- Disease Models, Animal MeSH
- Myocardium metabolism pathology MeSH
- Rats, Wistar MeSH
- Isolated Heart Preparation MeSH
- Ischemic Preconditioning methods MeSH
- Regional Blood Flow MeSH
- Myocardial Reperfusion Injury metabolism pathology prevention & control MeSH
- Mitochondria, Heart metabolism pathology MeSH
- Streptozocin MeSH
- Hindlimb blood supply MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Streptozocin MeSH
Various types of mechanosensitive ion channels, including cationic stretch-activated channels (SAC(NS)) and stretch-activated BKca (SAKca) channels, modulate heart rhythm. Bepridil has been used as an antiarrhythmic drug with multiple pharmacological effects; however, whether it is effective for mechanically induced arrhythmia has not been well investigated. To test the effects of Bepridil on SAKca channels activity, cultured chick embryonic ventricular myocytes were used for single-channel recordings. Bepridil significantly reduced the open probability of the SAKca channel (P(O)). Next, to test the effects of bepridil on stretch-induced extrasystoles (SIE), we used an isolated 2-week-old Langendorff-perfused chick heart. The left ventricle (LV) volume was rapidly changed, and the probability of SIE was calculated in the presence and absence of bepridil, and the effect of the drug was compared with that of Gadolinium (Gd(3+)). Bepridil decreased the probability of SIE despite its suppressive effects on SAKca channel activity. The effects of Gd(3+), which blocks both SAKca and SAC(NS), on the probability of SIE were the same as those of bepridil. Our results suggest that bepridil blocks not only SAKca channels but possible also blocks SAC(NS), and thus decreases the stretch-induced cation influx (stabilizing membrane potential) to compensate and override the effects of the decrease in outward SAKca current (destabilizing membrane potential).
- MeSH
- Anti-Arrhythmia Agents pharmacology therapeutic use MeSH
- Bepridil pharmacology therapeutic use MeSH
- Electrocardiography drug effects methods MeSH
- Myocytes, Cardiac drug effects physiology MeSH
- Ventricular Premature Complexes drug therapy physiopathology MeSH
- Cells, Cultured MeSH
- Chick Embryo MeSH
- Isolated Heart Preparation * MeSH
- Heart MeSH
- Large-Conductance Calcium-Activated Potassium Channels antagonists & inhibitors physiology MeSH
- Animals MeSH
- Check Tag
- Chick Embryo MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Anti-Arrhythmia Agents MeSH
- Bepridil MeSH
- Large-Conductance Calcium-Activated Potassium Channels MeSH
Hypertrophied hearts are known for increased risk of arrhythmias and are linked with reduced ischemic tolerance. However, still little is known about state characterized only by increased left ventricle (LV) mass fraction. Seventeen isolated rabbit hearts with various LV mass were divided into two groups according to LV weight/heart weight ratio (LVW/HW ratio), namely group H and L (with higher and lower LVW/HW ratio, respectively) and underwent three short cycles of global ischemia and reperfusion. The differences in electrogram (heart rate, QRS(max), mean number, onset and dominant form of ventricular premature beats) and in biochemical markers of myocardial injury (creatine kinase, lactate dehydrogenase - LDH) and lipid peroxidation (4-hydroxy-2-nonenal - 4-HNE) were studied. As compared to group L, hearts in group H exhibited lower tolerance to ischemia expressed as higher incidence and severity of arrhythmias in the first ischemic period as well as increase of LDH and 4-HNE after the first reperfusion. In the third cycle of ischemia-reperfusion, the preconditioning effect was observed in both electrophysiological parameters and LDH release in group H. Our results showed consistent trends when comparing changes in electrograms and biochemical markers. Moreover, 4-HNE seems to be good potential parameter of moderate membrane alteration following ischemia-reperfusion injury.
- MeSH
- Cardiac Complexes, Premature pathology physiopathology MeSH
- Hypertrophy, Left Ventricular pathology physiopathology MeSH
- Myocardial Ischemia pathology physiopathology MeSH
- Rabbits MeSH
- Isolated Heart Preparation methods MeSH
- Myocardial Reperfusion Injury pathology physiopathology MeSH
- Heart MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
