In Vitro Assessment of Cardiac Performance after Irradiation Using an Isolated Working Rat Heart Preparation

Summary

The effect of irradiation on cardiac function was assessed using an isolated working rat heart preparation. The animals were given single doses of X-rays in the range 15–30 Gy to their hearts. Cardiac output (CO = aortic flow + coronary flow), heart weight and body weight were followed for a period of 10 months after treatment. Irradiation led to a decrease in cardiac function. This reduction was dose-dependent and progressive with time after treatment. The shape of the Frank-Starling curves constructed for irradiated hearts suggests a loss of contractile function of the myocardium. Coronary flow rates measured in ‘working’ hearts and in ‘Langendorff’ hearts were not significantly changed by the irradiation treatment. The isolated working rat heart preparation proved to be a simple and suitable animal model for the investigation of irradiation-induced cardiotoxicity.     

MR imaging of coronary artery flow in isolated and in vivo hearts.

Methods for imaging flow in coronary arteries with magnetic resonance (MR) imaging techniques are demonstrated in isolated heart preparations and live animal models. Coronary artery flow was first imaged with a flow-compensated gradient-echo pulse sequence in isovolumic and working perfused rat hearts and then in vivo. A bolus tracking technique was used to measure flow velocity in the coronary arteries. Ultrafast gradient-echo imaging techniques were then applied, with high resolution obtained by combining the information from several cardiac cycles. A stimulated-echo pulse sequence was demonstrated as a method for performing coronary angiography by flow tagging in isovolumic perfused hearts. This report describes the results of coronary flow MR imaging in isolated rat hearts and live mice and rats. The general approach has proved useful in evaluating new methods for coronary MR angiography and should permit well-controlled studies of pathologic conditions. This ability to image coronary flow in isolated hearts and in small animals should permit integrated MR studies of coronary flow, myocardial perfusion, myocardial metabolism, and cellular ionic status.     

Proton electron double resonance imaging (PEDRI) of the isolated beating rat heart.

Proton electron double resonance imaging (PEDRI) is a double resonance technique where proton MRI is performed with irradiation of a paramagnetic solute. A low-field PEDRI system was developed at 20.1 mT suitable for imaging free radicals in biological samples. With a new small dual resonator, PEDRI was applied to image nitroxide free radicals in isolated beating rat hearts. Experiments with phantoms showed maximum image enhancement factors (IEF) of 42 or 28 with TEMPONE radical concentrations of 2-3 mM at EPR irradiation powers of 12W or 6W, respectively. In the latter case, image resolution better than 0.5 mm and radical sensitivity of 5 microM was obtained. For isolated heart studies, EPR irradiation power of 6W provided optimal compromise of modest sample heating with good SNR. Only a small increase in temperature of about 1 degrees C was observed, while cardiac function remained within 10% of control values. With infusion of 3 mM TEMPONE an IEF of 15 was observed enabling 2D or 3D images to be obtained in 27 sec or 4.5 min, respectively. These images visualized the change in radical distribution within the heart during infusion and clearance. Thus, PEDRI enables rapid and high-quality imaging of free radical uptake and clearance in perfused hearts and provides a useful technique for studying cardiac radical metabolism.     

Echo planar imaging of isolated perfused rat hearts at 4.7 T: a comparison of Langendorff and working heart preparations

The implementation of echo planar imaging at 4.7 T is demonstrated using a homebuilt gradient and radiofrequency assembly. The application of such a technique to the study of the isolated perfused rat heart is demonstrated. Langendorff and working heart perfusion preparations are compared and changes in the left ventricular volume shown are to be much larger in the working heart preparation. Such a methodology is expected to provide a useful model for the study of cardiac function and dynamics in the normal and diseased states under controlled perfusion conditions.     

离体心脏灌注模型的制备

目的 通过制备大鼠离体心脏灌注模型,探讨模型制备的方法及注意事项.方法 健康成年大鼠20只,雌雄不限,取离体心脏,制备Langendorff模型及离体工作心脏灌注模型.结果 实验中成功18例(成功率90%).心脏均能持续稳定搏动,心脏收缩有力,左室内压为7.1～10.5 kPa,处于生理范围(6.3～10.3 kPa),心率250～300 /min,心律齐,心脏可平稳跳动6 h以上.结论 Langendorff模型及离体工作心脏灌注模型操作方便、稳定,是比较符合生理特性的离体心脏灌注模型.     

Cardioprotective effect of spontaneous activity

In the perspective of giving a better understanding of the cardioprotective effects attributable to the tandem low caloric intake and training, Lou/C rats would be an interesting model since these animals exhibit spontaneously these two characteristics for months, without any dietary manipulations or stressor stimuli. No information was so far available on their cardiac function. Therefore, the aim of this pilot study was (i) to document cardiac function before and after ischemia in this strain, and (ii) to investigate whether spontaneous wheel-running activity can improve the ability of cardiac muscle to recover its function after an ischemic period. Cardiac mechanical and metabolic functions were measured in isolated Langendorff hearts from Wistar sedentary, Lou/C sedentary, and Lou/C wheel-running male rats submitted to a 20-min low-flow ischemia and 20-min reperfusion. In Lou/C sedentary rats, rate-pressure product, an index of cardiac work, was decreased before ischemia as compared to Wistar sedentary animals (- 24 %, p < 0.05). After ischemia, cardiac mechanical function recovery did not significantly differ between these two groups. Nevertheless, flux of non-oxidative glycolysis was lower before and after ischemia in Lou/C sedentary animals than in Wistar sedentary rats. In Lou/C rats, during normoxic perfusion, wheel-running activity significantly decreased heart rate (- 15 %), oxygen consumption (- 2.2 %) and cardiac efficiency (- 37 %), whereas coronary flow and flux of non-oxidative glycolysis were significantly increased (+ 15 % and + 263 %, respectively). After ischemia, recovery of cardiac mechanical function and cardiac efficiency were improved in Lou/C wheel-running rats versus Lou/C sedentary animals (p < 0.05). In conclusion, the impact of ischemia-reperfusion is similar between Lou/C- and Wistar sedentary rats. Spontaneous wheel-running activity decreases cardiac efficiency before ischemia and confers a protection against ischemia- and reperfusion-induced injury in isolated Lou/C rat hearts.     

Contractile function of the heart of guinea pigs after long-term continuous stress

Guinea-pigs exposed to 60- and 100-day biosocial stress were used to examine the contractile function of the heart in the whole body and in an isolated state. A 60-day stress effect caused adaptation which included increases in the intensity of heart contractility and in its weight unit by 29% and 38%, respectively. Measurement of resistance of such an adapted heart to isometric load produced by a short-term aortic occlusion showed increases in maximal rates of its contraction and relaxation by 34-35%. After a 100-day stress-effect the strength and velocity of heart contraction and resistance to isometric load returned to normal; however, the intensity of the function of the weight unit increased by 27-32%. The effect of adaptation was also seen in an isolated heart: after a 100-day exposure the isolated hearts did not lose their ability to react to a greater preload according to Starling's law; moreover at the filling pressure 15 cm H2O their cardiac output increased by 50% as compared to the baseline. This observation suggests that the process of adaptation of guinea-pig hearts to chronic stress involves not only activation of central mechanisms of regulation but also enhancement of mechanisms of self-regulation but also enhancement of mechanisms of self-regulation.     

Myocardial kinetics of 99m technetium-Q agents: studies in isolated cardiac myocyte, isolated perfused rat heart, and canine regional myocardial ischemia models.

OBJECTIVE: Based on reports of high cellular uptake and low plasma binding of nonreducible mixed ligand Tc(III) cations (Q complexes) and high linear uptake versus blood flow of 99mTc-Q3 in canine hearts, the authors hypothesized that the two Q complexes, 99mTc-Q63 and 99mTc-Q64, would have high cell uptake and better differentiation between ischemic and nonischemic myocardium compared with other 99mTc-based compounds. METHODS: Uptake and retention kinetics of 99mTc-Q63 and 99mTc-Q64 were measured in isolated rat cardiac myocytes, isolated perfused rat hearts, and intact canines and compared with previously reported Q-based compounds, a clinically available 99mTc perfusion agent (sestamibi), and 201Tl. RESULTS: Uptake of Q63, Q64, and sestamibi by isolated cardiac myocytes was similar. Maximum extraction (Emax) of Q64 by isolated perfused rat hearts was greatest among the 99mTc agents (P < 0.02), but net extraction (Enet) of Q64 was not different from Q63 or sestamibi 3 minutes after tracer injection. By 15 minutes, 201Tl Enet was lower than Q63, Q64, and sestamibi (P < 0.05). Among 99mTc agents, the uptake versus flow of Q3, Q63, and Q64 by canine heart was superior to Q12 and sestamibi (P < 0.05). CONCLUSIONS: The activity of Q63 and Q64 in the myocardium is related to actual myocardial blood flow over a broad, clinically relevant range of flows. The ischemic-to-normal zone activity distributions of Q63 and Q64 approximate actual flow in a manner more like that of 201Tl than sestamibi or Q12. These results provide a rational foundation in support of further evaluation of Q63 and Q64 in humans.     

Evaluation of cardiac beta-adrenoreceptors in the isolated perfused rat heart using (S)-11C-CGP12388.

(S)-(11)C-CGP12388 ((11)C-CGP12388) was recently developed as an in vivo PET tracer for the evaluation of cardiac beta-adrenergic receptors. The purpose of this study was to evaluate the myocardial kinetics of (11)C-CGP12388 using the perfused rat heart model. METHODS: Normal rat hearts were cannulated for retrograde perfusion according to the Langendorff method. Studies were performed using constant coronary flow rates of 12 mL/min (high flow: n = 6) and 6 mL/min (low flow: n = 6). Beta-adrenergic-blocking studies were also done using propranolol (blocking: n = 6). Two bolus injections of (11)C-CGP12388 were administered at a 25-min interval, and time-activity curves were measured using bismuth germanate detectors. The beta-adrenergic receptor density (B(max)) and total distribution volume (DV(tot)) were estimated using compartmental modeling. After the experiment, B(max) in vitro was measured for all hearts using (3)H-CGP12177, and the values were compared with the B(max) estimated in isolated hearts. RESULTS: DV(tot) was significantly lower in the blocking group than in the high-flow group (P < 0.01), and there was no significant difference in DV(tot) between the high- and the low-flow groups. B(max) values estimated from (11)C-CGP12388 kinetics were 5.05 +/- 0.90 pmol/g under the high-flow model and 5.20 +/- 0.63 pmol/g under the low-flow model. The B(max) results in isolated hearts correlated significantly with the measured in vitro B(max) values (r(2) = 0.69; P < 0.001). CONCLUSION: Beta-adrenoreceptor density in the isolated rat heart can be quantified using (11)C-CGP12388 and a 2-injection protocol. The binding of the tracer was flow independent, with low nonspecific binding. These results suggest that (11)C-CGP12388 is a promising PET tracer that may be applicable to human studies.     

Three-Dimensional gated EPR imaging of the beating heart: Time-resolved measurements of free radical distribution during the cardiac contractile cycle

In vivo or ex vivo EPR imaging, EPRI, has been established as a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. While instrumentation capable of performing EPR imaging of free radicals in whole tissues and isolated organs has been previously reported, it was not possible to image rapidly moving organs such as the beating heart Therefore instrumentation was developed to enable the performance of gated-spectroscopy and imaging on isolated beating rat hearts at L-band. A synchronized pulsing and timing system capable of gated acquisitions of up to 256 images per cycle, with rates of up to 16 Hz was developed. The temporal and spatial accuracy of this instrumentation was verified using a specially designed beating heart-shaped isovolumic phantom with electromechanically driven sinusoidal motion at a cycle rate of 5 Hz. Gated EPR imaging was performed on a series of isolated rat hearts perfused with nitroxide spin labels. These hearts were paced at a rate of 6 Hz with either 16 or 32 gated images acquired per cardiac contractile cycle. The images enabled visualization of the time-dependent alterations in the free radical distribution and anatomical structure of the heart that occur during the cardiac cycle.     

Gadolinium-DTPA-enhanced magnetic resonance imaging of the isolated rat heart after ischemia and reperfusion.

The objective of this study was to assess the potential of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) to identify myocardial ischemia and reperfusion in the isolated rat heart model. Ischemia was induced by reducing the perfusion pressure from 80 to 30 mm Hg for 2 hours. Hearts were not reperfused, or were reperfused for 20 minutes or for 2 hours. Perfusion was performed with Evans blue dye and/or Gd-DTPA for 3 minutes. Twenty isolated rat hearts were perfused according to the Langendorff method, and divided into five groups according to the perfusion status and the use of Gd-DTPA and/or Evans blue as perfusion markers. The Evans blue distribution in the hearts was assessed by point-counting volumetry. The Gd-DTPA distribution was assessed by magnetic resonance microimaging at 6.3 T field strength. Evans blue staining clearly identified areas with "no flow" or "no reflow." Perfusion with Gd-DTPA enhanced signal intensity significantly, both in ischemic and reperfused myocardium. Signal intensity in hearts reperfused for 2 hours was increased significantly compared to nonreperfused ischemic hearts, but not to ischemic hearts reperfused for 20 minutes. Magnetic resonance imaging with the aid of Gd-DTPA can identify ischemia and reperfusion in the isolated rat heart, dependent on residual perfusion.     

心脏保存液种类及保存时间对离体鼠心低温保存效果的对比研究

目的研究3种心脏保存液[HTK液、FWM液和St.Thomas-Ⅱ(ST-Ⅱ)]在低温条件下保存离体鼠心的效果。方法54只雄性SD大鼠随机均分为HTK组、FWM组、ST-Ⅱ组(n=18),各组再根据低温保存时间分为4、6、8h3个亚组(n=6)。采用Langen-dorff装置进行心脏保存前后的灌注,各实验组分别使用不同保存液灌停心脏并在低温条件下保存4、6、8h,于灌停前及复灌后测定心率(HR)、冠脉流量(CF)、左心室收缩压(LVSP)、左心室内压最大上升速率(+dp/dtmax)、左心室内压最大下降速率(-dp/dtmax)等指标,并计算各指标的恢复率。灌注结束后,取心肌组织制作光镜标本,观察心肌组织结构变化。结果在相同的低温保存时间下,与ST-Ⅱ组相比,HTK组与FWM组心功能各指标恢复率明显较高,光镜下心肌组织结构变化较小。随保存时间延长,使用HTK液保存的鼠心心功能恢复率下降,组织结构发生改变,8h亚组与4、6h亚组间比较差异显著(P<0.05),而4h亚组与6h亚组间无显著差异(P>0.05)。结论在保存时间相同的条件下,HTK液及FWM液的心肌保存效果明显优于ST-Ⅱ液。HTK液保存效果随低温...     

Facteurs de la variation de la fréquence cardiaque maximale: hypothèse «enképhalinique

To try to explain the decrease in maximal heart rate under different situations (ie, chronic hypoxia, ageing, proteino-energetic malnutrition), cardiac concentration and flow of leucine-encephalin peptide in isolated and perfused hearts were studied in rats. The heart leucine-encephalin concentration which was 301 ± 60 fmol/g before perfusion, increased up to 543 ± 125 fmol/g after perfusion (1 h). The total release during perfusion was four times the initial concentration. Concerning the antagonism of leucine-encephalin on cardiotropic adrenergic effects on isolated cardiomyocytes, it is possible to propose this mechanism to explain totally or partially the decrease in maximal heart rate under particular situations.     

The effects of 17 alpha-methyltestosterone on myocardial function in vitro

Testosterone analogs have been used as performance enhancers by athletes for more than 40 yr. We asked whether the anabolic steroid 17 alpha-methyl-4-androstene-17-ol-3-one (17 alpha-MT) would affect intrinsic contractile function of the heart. Male Sprague-Dawley rats, 125-150 g, were treated with 17 alpha-MT either parenterally or orally for up to 8 wk. Intrinsic contractile function of the hearts was assessed utilizing both the isolated working heart and isovolumic perfused heart preparations. Isolated working hearts from 17 alpha-MT-treated rats had a 45% decrease in heart work attributable largely to a similarly decreased stroke volume. Isovolumic perfused hearts from treated animals had elevated left ventricular systolic and diastolic pressures at similar interventricular volumes compared to controls. Rates of ventricular pressure development (+dP/dT) or relaxation (-dP/dT) were unchanged as a result of the treatment. However, static elastance was reduced in potassium-arrested hearts from the 17 alpha-MT treatment (63% increase in interventricular pressure), consistent with a limitation being imposed on stroke volume by a decreased myocardial compliance. Hydroxyproline content of the hearts was not altered by 17 alpha-MT treatment suggesting that increased stiffness was not a consequence of collagen proliferation. Treatment of the steroid rats with beta-aminopropionitrile, a compound that inhibits lysyl oxidase, restored the left ventricular volume-pressure relationship (elastance curve) to that of control hearts. Thus, chronic treatment with anabolic steroids appears to reduce left ventricular compliance, possibly related to an enhanced activity of lysyl oxidase, and results in increased crosslink formation between collagen strands in the extracellular matrix.     

低G预适应对重复+10Gz应激大鼠左心室收缩功能和心肌内皮素与前列环素含量的影响

目的 进一步验证低G预适应对 Gz应激致心肌损伤的防护效应。方法 42只雄性Wistar大鼠随机分为3组(每组n=14)：A( 1Gz)组、B( 10Cz)组和C(低G预适应)组。B组实验条件为： 10Gz峰值暴露30s，5次／d(间隔 1Gz 1min)，3(1／wk，共3wk；A组仅受 1Gz作用5min；C组在每日 10Cz暴露前约1h受 2Gz作用5min，其余处理同B组。于末次 Gz实验后次日断头处死大鼠，速取心脏离体观察左室收缩压的变化；另取心肌组织测定其内皮素与前列环素含量。结果与A组相比，B组心肌收缩功能明显降低，心肌内皮素含量显著升高；而低G预适应使 10Gz应激大鼠左室收缩压明显增加，心肌内皮素含量明显降低。结论 低G预适应对重复高G应激大鼠左室功能和心肌血管内皮细胞分泌功能均有一定的保护作用。     

HBOC attenuates intense exercise-induced cardiac dysfunction

The purpose of this study was to investigate whether hemoglobin-based oxygen carrier (HBOC) could protect the heart from intense exercise-induced myocardial dysfunction. Adult male Sprague-Dawley rats were subjected to 5-h intense prolonged running on treadmill with or without HBOC pre-treatment. Immediately after exercise, the heart rate (HR) and oxygen delivery capacity of the blood were measured. After 1 h of rest, echocardiography was performed to assess the post-exercise cardiac function. Then all the hearts were isolated and perfused using the Langendorff model for 1 h. Our results proved that pronged exercise caused significant LV dysfunction, while HBOC pre-treatment attenuated such a damage, as evidenced by the increased oxygen delivery, cardiac fractional shortening (FS), rate-pressure product (RPP), ± dp/dt and coronary flow rate (CF) and decreased myocardial necrosis. The releases of cardiac enzymes, including creatine kinase-MB (CK-MB) and cardiac troponin-I (cTnI) were markedly reduced. No significant difference of cardiac infarct size was observed among groups. In addition, HBOC significantly elevated superoxide dismutase (SOD) activity and decreased hydrogen peroxide (H2O2) formation, which indicated the exercise-induced cardiac oxidative damage was inhibited. In conclusion, HBOC pre-treatment showed a promising cardioprotective effect on prolonged exercise-induced cardiac dysfunction, which was probably associated with its ability to decrease myocardium oxidative stress.     

Effect of myocardial perfusion and metabolic interventions on cardiac kinetics of phenylpentadecanoic acid (IPPA) I 123

The effect of regional myocardial perfusion and flow-independent adrenergic stimulation, as well as lactatemediated inhibition of cardiac lipolysis, on cardiac IPPA uptake and metabolism was examined in canine hearts (flow studies) and in the isolated perfused Langendorff rat heart (metabolic interventions). In both normal and ischaemic myocardium, local perfusion is a major determinant of cardiac IPPA uptake. In pacing-induced hyperaemia, the strict flow-dependence of cardiac IPPA uptake is not preserved. Adrenergic stimulation raises the rate of oxidation of both palmitic acid ¹⁴C and IPPA. This change is reflected by increased metabolite production released into the perfusate and radioactivity clearance recorded externally. Lactate in high concentrations exerts the opposite effect on cardiac free fatty acid oxidation. IPPA is stored in this condition preferentially in tissue phospholipids and triglycerides.

     

Intracellular Volume Measurement and Detection of Edema: Multinuclear NMR Studies of Intact Rat Hearts during Normothermic Ischemia

The present study describes the cell volume dynamics in intact rat hearts, during ischemia and after reperfusion. Cell volumes were measured in isolated hearts by either ¹³C or ⁵⁹Co NMR of mannitol or cobalticyanide, respectively, as extracellular markers and ¹H NMR of water as the aqueous space marker. A constant volume chamber was built inside a 15-mm NMR tube; the contents of the chamber were measured with and without a heart. The intracellular volume of isolated rat hearts was estimated to be 2.45 ± 0.13 ml/g dry weight. In the perfused heart, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations were calculated to be 12.2 ± 0.7 and 16.1 ± 1.0 mM, respectively. Consecutive volume measurements showed cell swelling of 16% during 30 min of ischemia, which was reduced at reperfusion to 7%. After 30 min of reperfusion, ATP and PCr concentrations were 4.5 ± 0.8 and 8.1 ± 0.9 mM. It is concluded that: (1) cell swelling is an ischemic event, which is partially reversed by reperfusion; and (2) continuous measurement of cell volumes provides intracellular molar concentrations of metabolites, which are the physiologically significant parameters.     

茶多酚对重复+Gz暴露大鼠心功能和心肌超微结构的影响

目的观察天然抗氧化剂茶多酚对重复＋１０Ｇｚ暴露大鼠心功能和心肌超微结构的影响。方法２４只雄性Ｗｉｓｔａｒ大鼠随机等分为三组：Ａ组,Ｂ组,Ｃ组,Ｂ,Ｃ组受重复＋１０Ｇｚ作用,而Ａ组仅受＋１Ｇｚ作用。Ｃ组于＋Ｇｚ实验前约１ｈ灌胃给予ＴＰ,另两组给予等量蒸馏水。测定离体大鼠工作心脏的心功能并用电镜观察心肌超微结构的变化。结果与对照组相比,重复＋１０Ｇｚ暴露使大鼠左室内压峰值明显降低,心肌细胞超微结构出现     

静磁场辐射对大鼠心脏功能和结构的影响研究

目的研究静磁场辐射对大鼠心脏生理功能与组织结构的影响,为静磁场辐射损伤机制研究奠定基础。方法将20只Wistar雄性大鼠置于600 mT的静磁场中,每天辐射12 h,连续7 d。大鼠于辐射后7 d和14 d检测心率和心电图变化后活杀,取心肌组织入10%甲醛缓冲溶液固定、石蜡包埋、HE染色,光镜观察心脏组织结构改变。结果 600 mT静磁场辐射后7 d,大鼠心电图显示心率显著下降（P〈0.01）,P波时程以及T-H较对照组显著升高（P〈0.05或0.01）;辐射后14 d,大鼠心率以及R-H较辐射前显著下降（P〈0.01）,而T-H较辐射前显著升高（P〈0.05）。600 mT静磁场辐射后7 d,大鼠室间隔及心内膜下心肌细胞嗜酸性变、肌纤维凝聚,核固缩;心肌纤维呈波浪状排列;血管扩张、淤血,管腔内见血浆蛋白和红细胞混杂,示血栓形成;辐射后14 d,上述病变进一步加重。结论 600 mT静磁场辐射可对大鼠心脏功能和结构造成损伤,表现为心电图异常、心脏血液循环障碍,心肌细胞变性和坏死,并在辐射后2周内呈加重趋势。