NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff¹, the isolated perfused heart remains a prominent tool for studying cardiac physiology². However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures³. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism^4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model^{7, 8}. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode⁸. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours⁸.When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)^9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration¹² and provides a measure of mitochondrial redox state¹³. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions^{14, 15} and to monitor the progression of hypoxic conditions over time¹⁰.The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution¹⁶ at 37 °C. Aortic, LV, pulmonary artery, and left & right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350±25 nm) and used to illuminate the epicardium. Emitted light was filtered (460±20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.     

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.     

Function and energy metabolism of isolated hearts obtained from hyperthyroid spontaneously hypertensive rats (SHR)

It was the aim of this study to evaluate the effects of hyperthyroidism on heart function and cardiac energy metabolism of spontaneously hypertensive (SHR) rats. Hyperthyroidism was induced by daily injections of T₃ (0.2 mg/kg s.c.) for 14 days. The hearts were then isolated and perfused in the Langendorff mode. ATP, phosphocreatine (PCr), and inorganic phosphate (Pi) were measured continuously by means of³¹P-nuclear magnetic resonance (NMR) spectroscopy. Work load was altered by varying stepwise the Ca⁺⁺ concentration in the perfusion fluid from 0.5 to 1.0, 1.5, and 2.0 mM, respectively. At every elevation of the Ca⁺⁺ concentration, the increase in left ventricular developed pressure (LVDP) was higher in the hyperthyroid SHR than in the untreated SHR hearts. The ATP and PCr concentrations were lower in the hyperthyroid SHR compared to the untreated SHR hearts throughout the perfusion period. PCr decreased at every Ca⁺⁺ elevation in both the untreated and hyperthyroid SHR hearts. The PCr/ATP ratio was not altered at any Ca⁺⁺ concentration neither in the untreated SHR nor in the hyperthyroid SHR hearts. The Ca⁺⁺-induced stepwise elevation in LVDP was higher at any given PCr/Pi ratio in the hyperthyroid SHR than in the untreated SHR hearts. Thus, the Ca⁺⁺-inducible contractile reserve was greater in the hyperthyroid SHR heart.     

成年小鼠心肌细胞分离技术

为进行成年小鼠心肌细胞培养与收缩功能研究, 首先必须获得高产量与高质量的心肌细胞。本实验采用Langendorff装置行恒流灌流心脏, 同时监测灌流压力的变化。根据小鼠鼠龄微调灌流流速, 使初始灌流压力保持在40 mmHg。用0.05 % 单一粗制胶原酶在37 ℃条件下消化心脏, 当灌流压力下降至28 mmHg 时, 即刻终止消化。轻轻吹散心肌细胞后, 用含1 % 牛血清白蛋白的 Joklik’s MEM 培养液保存,逐步法恢复细胞外钙离子浓度。获得的心肌细胞存活率大于 70 %,复钙后耐钙心肌细胞静置 4 h,心肌细胞存活率仍能保持在(40~50) %。其中,90 %以上存活的长杆状心肌细胞无明显搏动,细胞膜表面光滑,横纹清晰,两端边缘锐利, 折光性较强, 复钙后保存4 h 或 5.0 Hz 刺激5 min 后,仍能保持正常形态。在1.0 Hz刺激条件下, 心肌细胞缩短幅度为(9.72 ±0.43) %; 2.0 Hz 刺激下为(11.28 ±0.43) %; 在5.0 Hz 刺激下, 达到(11.40 ±0.45)%。这些结果表明, 采用本方法可获得高产量与高质量的成年小鼠心肌细胞, 且易于操作, 重复性较好。     

Change in substrate metabolism in isolated mouse hearts following ischemia-reperfusion

Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using ³H-glucose, ¹⁴C-glucose and ³H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat.     

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).     

Catheter-mediated subselective intracoronary gene delivery to the rabbit heart: introduction of a novel method

BACKGROUND: Recent studies suggest that gene therapy using replication-deficient adenoviruses will benefit treatment of cardiovascular diseases including heart failure. A persistent hurdle is the effective and reproducible delivery of a transgene to the myocardium with minimal iatrogenic morbidity. In this study, we sought to design a relatively non-invasive percutaneous gene delivery system that would maximize cardiac transgene expression and minimize mortality after intracoronary adenovirus injection. METHODS: Adult rabbits received a left circumflex coronary artery (LCx) infusion of 5x10(11) total viral particles of an adenovirus containing the marker transgene beta-galactosidase (Adeno-betaGal) via either a continuous infusion method utilizing an oxygenated, normothermic, physiologic pH Krebs solution driven by a Langendorff apparatus (n=12) or a timed bolus and set concentration at a constant infusion rate to the LCx (n=12). Six rabbits underwent global transgene delivery via an invasive method involving intraventricular delivery and aortic root cross-clamping. The efficacy of transgene expression via these three distinct delivery methods was determined in the left ventricle at 5 days by histological staining and colorimetric quantification assay. RESULTS: While the open-chest, aortic cross-clamping method provides the highest level of gene expression throughout the heart, the morbidity of this procedure is clinically prohibitive. Percutaneous LCx delivery of Adeno-betaGal using the Langendorff apparatus was associated with the lowest morbidity and mortality while still supporting significant myocardial gene expression. CONCLUSIONS: Percutaneous delivery of an adenovirus solution using a continuous infusion of oxygenated Krebs solution via a Langendorff apparatus appears to be a gene delivery modality offering the best compromise of gene expression and clinical utility to maximize any potential therapeutic outcome.     

Phospholamban S-nitrosylation modulates Starling response in fish heart

The Frank–Starling mechanism is a fundamental property of the vertebrate heart, which allows the myocardium to respond to increased filling pressure with a more vigorous contraction of its lengthened fibres. In mammals, myocardial stretch increases cardiac nitric oxide (NO) release from both vascular endothelium and cardiomyocytes. This facilitates myocardial relaxation and ventricular diastolic distensibility, thus influencing the Frank–Starling mechanism.In the in vitro working heart of the eel Anguilla anguilla, we previously showed that an endogenous NO release affects the Frank–Starling response making the heart more sensitive to preload. Using the same bioassay, we now demonstrate that this effect is confirmed in the presence of the exogenous NO donor S-nitroso-N-acetyl penicillamine, is independent from endocardial endothelium and guanylate cyclase/cGMP/protein kinase G and cAMP/protein kinase A pathways, involves a PI(3)kinase-mediated activation of endothelial NO synthase and a modulation of the SR-CA²⁺ATPase (SERCA2a) pumps. Furthermore, we show that NO influences cardiac response to preload through S-nitrosylation of phospholamban and consequent activation of SERCA2a. This suggests that in the fish heart NO modulates the Frank–Starling response through a beat-to-beat regulation of calcium reuptake and thus of myocardial relaxation.We propose that this mechanism represents an important evolutionary step for the stretch-induced intrinsic regulation of the vertebrate heart, providing, at the same time, a stimulus for mammalian-oriented studies.     

Cardiac dynamics of the Langendorff perfused heart

The Langendorff perfused heart is studied in a closed system with (i) automatic regulations to maintain constancy of the perfusion column (Krebs-Henseleit + 0.5% albumin or 25-30% washed erythrocyte suspension), (ii) continuous recording of rate, coronary flow, and supravalvular aortic pressure. A microcomputer with software interface is used for storage treatment and on-line analysis of the recorded variables. In 38 preparations perfused with Krebs-Henseleit, minimal diastolic (61.2 +/- 2.8 mm Hg) is significantly below and peak systolic (98.7 +/- 3.6 mm Hg) significantly above perfusion pressure (80 mm Hg). Pressure difference between minimal diastolic and peak systolic (delta P) is 37.5 +/- 1.8 mm Hg. Increases in perfusion pressure will be associated with increases of coronary flow and delta P, which is also increased by isoprenaline administration. Oxygen consumption decreased by 76% when perfusion pressure was lowered from 80 to 60 mm Hg in hearts perfused with a 30% erythrocyte suspension. All of these experimental results were interpreted as indicating that delta P measured in this system resulted from an ejected volume (x acceleration) from the heart. The ejected volume corresponds to a valvular leak caused by the rigid nature of the system which is devoid of aortic compliance. delta P may be considered an index of left ventricular performance, an indication that the Langendorff preparation studied under the present conditions is a working heart. A 100-microliter volume constant infusion syringe for time administration of cardioactive drugs may be inserted at the base of the perfusion column to obtain dose-response effects.     

Effects of prostacyclin on coronary circulation, heart rate and myocardial contractile force in isolated hearts of guinea PIG and rabbit — Comparison with prostaglandin E2

Infusions of prostacyclin (PGI₂) (3 × 10⁻¹⁰ − 3 × 10⁻⁷M) into the coronary circulation of isolated hearts from guinea pigs or rabbits resulted in a concentration-dependent decrease in the coronary perfusion pressure (CPP). There was a slight decrease in left ventricular systolic pressure in the heart of the rabbit, whereas the heart rate remained unchanged. PGE₂ was without effect on the heart of the rabbit but was as potent as PGI₂ in decreasing the CPP in the guinea pig heart. 6-oxo-PGF_1α (up to 3 × 10⁻⁶ M) did not affect any of the parameters measured.     

Effects of glyburide (glibenclamide) on myocardial function in Langendorff perfused rabbit heart and on myocardial contractility and slow calcium current in guinea-pig single myocytes

Glyburide, also known as glibenclamide, was shown to have positive inotropic effect in human and animal hearts. The objectives of the present study was to investigate the effects of glyburide on developed left ventricular pressure (DLVP), coronary flow (CF), and heart rate (HR), in isolated rabbit heart as well as its effects on myocardial contractility and L-type calcium current, i_Ca, in guinea pig myocytes. Rabbit hearts were mounted on Langendorff apparatus and perfused with an oxygenated Krebs for 30 min until reaching steady state to be followed by 20 min of experimental perfusion divided into 5 min of control perfusion and 15 min of perfusion with Glyburide (10 M). Ventricular myocytes were isolated by enzymatic dispersion technique and superfused in an oxygenated Tyrode solution. Cells were voltage-clamped at holding potential –40 mV to inactivate Na⁺ current and a step depolarizations, 200 msec duration, to 0 mV was applied to elicit i_Ca. The contractions of the myocytes were measured by optical methods. Glyburide significantly increased DLVP by 30% and CF by 36% but had no effect on HR. Glyburide increased cell contractility by 7 ± 6, 18 ± 7, 28 ± 9 and 54 ± 15% for 0.1, 1, 10 and 100 M respectively, p < 0.001. Meanwhile it depressed i_Ca by 9 ± 6 and 19 ± 8% for 1 and 10 M respectively. In conclusion, glyburide increased contractility of guinea pig single myocytes and of isolated rabbit heart, as indicated by increased developed left ventricular pressure while it depressed i_Ca. It is hypothesized that an elevation in intracellular calcium, which caused increased myocardial contractility, could be attributed to an increase in intracellular Na⁺ that could increase intracellular calcium via Na⁺/Ca²⁺ exchange.     

The low oxygen-carrying capacity of Krebs buffer causes a doubling in ventricular wall thickness in the isolated heart

The buffer-perfused Langendorff heart is significantly vasodilated compared with the in vivo heart. In this study, we employed ultrasound to determine if this vasodilation translated into changes in left ventricular wall thickness (LVWT), and if this effect persisted when these hearts were switched to the "working" mode. To investigate the effects of perfusion pressure, vascular tone, and oxygen availability on cardiac dimensions, we perfused hearts (from male Wistar rats) in the Langendorff mode at 80, 60, and 40 cm H2O pressure, and infused further groups of hearts with either the vasoconstrictor endothelin-1 (ET-1) or the blood substitute FC-43. Buffer perfusion induced a doubling in diastolic LVWT compared with the same hearts in vivo (5.4 +/- 0.2 mm vs. 2.6 +/- 0.2 mm, p < 0.05) that was not reversed by switching hearts to "working" mode. Perfusion pressures of 60 and 40 cm H2O resulted in an increase in diastolic LVWT. ET-1 infusion caused a dose-dependent decrease in diastolic LVWT (6.6 +/- 0.4 to 4.8 +/- 0.4 mm at a concentration of 10(-9) mol/L, p < 0.05), with a concurrent decrease in coronary flow. FC-43 decreased diastolic LVWT from 6.7 +/- 0.5 to 3.8 +/- 0.7 mm (p < 0.05), with coronary flow falling from 16.1 +/- 0.4 to 8.1 +/- 0.4 mL/min (p < 0.05). We conclude that the increased diastolic LVWT observed in buffer-perfused hearts is due to vasodilation induced by the low oxygen-carrying capacity of buffer compared with blood in vivo, and that the inotropic effect of ET-1 in the Langendorff heart may be the result of a reversal of this wall thickening. The implications of these findings are discussed.     

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions³, monitor drug delivery^4,5, and encapsulation⁴. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.⁶ Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)⁷. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking⁸. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions³. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest⁹. Drug encapsulation can be achieved as reported during liposome formation⁹. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.Nonetheless, the current liposome preparation technologies (sonication¹⁰, freeze-and-thaw¹⁰, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency^10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.Manual extrusion using gas-tight syringes and polycarbonate membranes^10,5 is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)¹⁰, electron microscopy¹¹ and nanoparticle tracking analysis (NTA)¹² were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.     

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches¹. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible¹. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization¹. The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart². It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling³. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart^4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy^8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view^10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia^14-16.     

Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (;working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [(14)C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and (14)CO(2)). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue contents of hexose phosphates and of citrate. There were similar findings when working hearts from streptozotocin-diabetic rats with insulin added to the medium were compared with normal hearts. 8. The effects of insulin addition or of the chronic diabetic state could be explained in terms of an action of insulin on glucose transport. Increased heart work also acted at this site, but in addition there was evidence for altered regulation of glycolysis mediated by changes in tissue contents of adenine nucleotides or of citrate.     

Characterization of oxygen radical formation mechanism at early cardiac ischemia

Myocardial ischemia–reperfusion (I/R) causes severe cardiac damage. Although the primary function of oxymyoglobin (Mb) has been considered to be cellular O₂ storage and supply, previous research has suggested that Mb is a potentially protective element against I/R injury. However, the mechanism of its protective action is still largely unknown. With a real-time fluorescent technique, we observed that at the onset of ischemia, there was a small burst of superoxide (O₂^•–) release, as visualized in an isolated rat heart. Thus, we hypothesize that the formation of O₂^•– correlates to Mb due to a decrease in oxygen tension in the myocardium. Measurement of O₂^•– production in a Langendorff apparatus was performed using surface fluorometry. An increase in fluorescence was observed during the onset of ischemia in hearts perfused with a solution of hydroethidine, a fluorescent dye sensitive to intracellular O₂^•–. The increase of fluorescence in the ischemic heart was abolished by a superoxide dismutase mimic, carbon monoxide, or by Mb-knockout gene technology. Furthermore, we identified that O₂^•– was not generated from the intracellular endothelium but from the myocytes, which are a rich source of Mb. These results suggest that during the onset of ischemia, Mb is responsible for generating O₂^•–. This novel mechanism may shed light on the protective role of Mb in I/R injury.     

Transient changes in cyclic AMP and in the enzymic activity of protein kinase and phosphorylase during the cardiac cycle in the canine myocardium and the effect of propranolol

Summary Oscillation of cyclic AMP and in the activity ratio of cyclic AMP-dependent protein kinase and of glycogen phosphorylase with the cardiac cycle were demonstrated in the canine heart in situ. For tissue sampling an ECG (R-wave)-triggered, automatically working push-freeze-drill apparatus was developed which allows intraventricular cryobiopsies from the left ventricular muscle of anaesthetized open-chest dogs. The nucleotide cyclic AMP oscillated with the cardiac cycle during normal working condition, the higher cyclic AMP level occuring during systole. Cyclic GMP was assayed to be without oscillatory changes during the contraction-relaxation cycle. The rise in the activity ratio of protein kinase was found to coincide with the maximum in the level of cyclic AMP. Propranolol pretreatment prevents the transient in the level of the nucleotide as well as in the activity ratio of the kinase indicating i) a causal relationship between these changes and ii) a neurohumoral, beat-to-beat regulation by catecholamines released from the sympathetic nerve endings within the heart. Contrary the activity ratio of phosphorylase retains its transient changes during the cardiac cycle in the presence of propranolol, indicating a Ca-mediated activation of phosphorlase kinase during the contraction process.