Isolated Beating Rat Hearts Research Articles

Coronary Flow Assessment Using 3-Dimensional Ultrafast Ultrasound Localization Microscopy

BackgroundDirect assessment of the coronary microcirculation has long been hampered by the limited spatial and temporal resolutions of cardiac imaging modalities. ObjectivesThe purpose of this study was to demonstrate 3-dimensional (3D) coronary ultrasound localization microscopy (CorULM) of the whole heart beyond the acoustic diffraction limit (<20 μm resolution) at ultrafast frame rate (>1000 images/s). MethodsCorULM was performed in isolated beating rat hearts (N = 6) with ultrasound contrast agents (Sonovue, Bracco), using an ultrasonic matrix transducer connected to a high channel–count ultrafast electronics. We assessed the 3D coronary microvascular anatomy, flow velocity, and flow rate of beating hearts under normal conditions, during vasodilator adenosine infusion, and during coronary occlusion. The coronary vasculature was compared with micro-computed tomography performed on the fixed heart. In vivo transthoracic CorULM was eventually assessed on anaesthetized rats (N = 3). ResultsCorULM enables the 3D visualization of the coronary vasculature in beating hearts at a scale down to microvascular structures (<20 μm resolution). Absolute flow velocity estimates range from 10 mm/s in tiny arterioles up to more than 300 mm/s in large arteries. Fitting to a power law, the flow rate–radius relationship provides an exponent of 2.61 (r2 = 0.96; P < 0.001), which is consistent with theoretical predictions and experimental validations of scaling laws in vascular trees. A 2-fold increase of the microvascular coronary flow rate is found in response to adenosine, which is in good agreement with the overall perfusion flow rate measured in the aorta (control measurement) that increased from 8.80 ± 1.03 mL/min to 16.54 ± 2.35 mL/min (P < 0.001). The feasibility of CorULM was demonstrated in vivo for N = 3 rats. ConclusionsCorULM provides unprecedented insights into the anatomy and function of coronary arteries at the microvasculature level in beating hearts. This new technology is highly translational and has the potential to become a major tool for the clinical investigation of the coronary microcirculation.

3D ultrasound coronarography: first proof of concept study on isolated beating rat hearts

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Bettencourt Foundation Background/Introduction We demonstrated recently that Ultrafast ultrasound Doppler imaging can image the intramyocardial coronary circulation in beating hearts of large animals and patients [1]. Yet, ultrasound spatial resolution remains limited by wave physics and coronaries smaller than ∼100 µm could not be imaged. Ultrasound Localization Microscopy (ULM) [2] was recently introduced to tackle this issue and exploit the micrometric localization of microbubble contrast agents at ultrafast frame rate in order to image blood flows in micrometer vessels. Purpose The objective of this work was to demonstrate that 3D ultrafast ultrasound with contrast agents can provide the full 3D mapping of the coronary microcirculation with quantitative flow velocity on a beating rat heart. Methods Acquisitions were performed on ex vivo rat hearts (n = 5) with retrograde perfusion (Langendorff model). A flow of a Krebs–Henseleit solution mixed with a diluted microbubbles solution (0.22%) was perfused at controlled pressure into the coronary arteries (between 5 and 15 mL/min). We used a 32 × 32 elements, 8-MHz matrix-array ultrasound transducer connected to a 1024-channel programmable ultrasound scanner. An ultrafast Doppler imaging sequence consisting of 9 plane waves was transmitted at a PRF of 20 kHz during 270 ms and repeated 40 times. After beamforming and SVD clutter filtering, the microbubbles were localized and tracked in 3D. Flow velocity were mapped at baseline and after infusion of Adenosine (10e-5 µMol) at constant coronary perfusion pressure (120 mm Hg). Eventually, the hearts were fixed using formaldehyde perfusion and imaged by µCT after injection of radio opaque agent. Results We successfully imaged the coronary blood flows of entire rat hearts. It revealed the entire vasculature from large main coronaries arteries (cross section up to 1 mm) to small arterioles (smaller than 40 µm). Coronary flow velocities ranged from [1 – 50] cm/s depending on the arteries diameter. Velocity estimates were validated in vitro in tubes of Ø0.58mm and were in good agreement with theoretical values of a Poiseuille’s flow (relative ratio of 10% for maximum velocities). After Adenosine infusion, perfusion flow rates increased 102% ± 50% (p &amp;lt; 0.05) on average. Eventually, anatomy revealed by 3D ultrasound coronarography was in accordance with the anatomy revealed by the µCT. Conclusion(s) We demonstrated the feasibility of 3D ultrasound coronarography on isolated beating rat hearts. This technique has the potential to become a novel imaging tool to investigate the coronary micro-circulation and quantify non-invasively the Coronary Flow Reserve (CFR). Abstract Figure. Ultrasound coronarography

Induction of chagasic-like arrhythmias in the isolated beating hearts of healthy rats perfused with Trypanosoma cruzi-conditioned medium

Chagas' myocardiopathy, caused by the intracellular protozoan Trypanosoma cruzi, is characterized by microvascular alterations, heart failure and arrhythmias. Ischemia and arrythmogenesis have been attributed to proteins shed by the parasite, although this has not been fully demonstrated. The aim of the present investigation was to study the effect of substances shed by T. cruzi on ischemia/reperfusion-induced arrhythmias. We performed a triple ischemia-reperfusion (I/R) protocol whereby the isolated beating rat hearts were perfused with either Vero-control or Vero T. cruzi-infected conditioned medium during the different stages of ischemia and subsequently reperfused with Tyrode's solution. ECG and heart rate were recorded during the entire experiment. We observed that triple I/R-induced bradycardia was associated with the generation of auricular-ventricular blockade during ischemia and non-sustained nodal and ventricular tachycardia during reperfusion. Interestingly, perfusion with Vero-infected medium produced a delay in the reperfusion-induced recovery of heart rate, increased the frequency of tachycardic events and induced ventricular fibrillation. These results suggest that the presence of parasite-shed substances in conditioned media enhances the arrhythmogenic effects that occur during the I/R protocol.

Induction of chagasic-like arrhythmias in the isolated beating hearts of healthy rats perfused with Trypanosoma cruzi-conditioned medium

Chagas' myocardiopathy, caused by the intracellular protozoan Trypanosoma cruzi, is characterized by microvascular alterations, heart failure and arrhythmias. Ischemia and arrythmogenesis have been attributed to proteins shed by the parasite, although this has not been fully demonstrated. The aim of the present investigation was to study the effect of substances shed by T. cruzi on ischemia/reperfusion-induced arrhythmias. We performed a triple ischemia-reperfusion (I/R) protocol whereby the isolated beating rat hearts were perfused with either Vero-control or Vero T. cruzi-infected conditioned medium during the different stages of ischemia and subsequently reperfused with Tyrode's solution. ECG and heart rate were recorded during the entire experiment. We observed that triple I/R-induced bradycardia was associated with the generation of auricular-ventricular blockade during ischemia and non-sustained nodal and ventricular tachycardia during reperfusion. Interestingly, perfusion with Vero-infected medium produced a delay in the reperfusion-induced recovery of heart rate, increased the frequency of tachycardic events and induced ventricular fibrillation. These results suggest that the presence of parasite-shed substances in conditioned media enhances the arrhythmogenic effects that occur during the I/R protocol.

Effects of low-level lead exposure on blood pressure and function of the rat isolated heart

Objective:Exposure to low levels of lead acetate can induce hypertension in both humans and experimental animals. The exact mechanisms of lead-induced hypertension are not well understood, but its pathogenesis could be explained by the changes in heart rate and contractility.Materials and Methods:In the present study, the effects of exposure to 100 ppm of lead in drinking water (for periods of 4, 8, and 12 weeks) on blood pressure and some physiologic parameters (eg, electrocardiography [ECG], heart rate [HR], cardiac contractility, and coronary flow) of isolated beating rat heart was investigated using the Langendorff isolated heart apparatus. The isolated hearts were perfused with Krebs-Henseleit solution (37°C; pH 7.4; gassed with 95% O2 + 5% CO2). All data were digitized by a software program for further analysis.Results:The blood pressure in the 8- and 12-week lead-exposed groups was significantly increased as compared to the control group. The ECG showed arrhythmias and conduction abnormalities only in the late phases of exposure (12 weeks). The HR and contractility were significantly higher in the 8- and 12-week lead-treated rats but not in the 4-week group. No significant changes were observed in coronary flow.Conclusion:These results indicate that: 1) low levels of lead exposure do not significantly affect the ECG in the early phase, 2) low levels of lead exposure causes ECG changes in the late phases of exposure, and 3) this level of lead exposure can increase HR and cardiac contractility but has no effect on coronary flow.

Transverse oriented electric field re-entrant resonator (TERR) with automatic tuning and coupling control for EPR spectroscopy and imaging of the beating heart

Sample motion, particularly that of a beating heart, induces baseline noise and spectral distortion on an EPR spectrum. In order to quench motional noise and restore the EPR signal amplitude and line-width, an L-band transverse oriented electric field re-entrant resonator (TERR) was designed and constructed with provisions for automatic tuning control (ATC) and automatic coupling control (ACC) suited for studies of isolated beating rat hearts. Two sets of electronic circuits providing DC biased voltage to two varactor diodes were implemented to electronically adjust coupling and tuning. The resonator has a rectangular cross-sectional sample arm of 25 mm diameter with a Q value of 1100 without sample. Once inserted with lossy aqueous samples of 0.45% NaCl, Q value drops to 400 with a volume of 0.5 ml and 150 with 5 ml. The ATC/ACC functions were tested with a moving phantom and isolated beating rat hearts with the improvement of signal to noise ratio (S/N, peak amplitude of signal over peak amplitude of baseline noise) of 6.7-, and 4 to 6-fold, respectively. With these improvements, EPR imaging could be performed on an isolated beating rat heart. Thus, this TERR resonator with ATC/ACC enables application of EPR spectroscopy and imaging for the measurement and imaging of radical metabolism, redox state, and oxygenation in the isolated beating rat heart.

Role of Nitric Oxide and Mitochondrial Nitric Oxide Synthase in Energy Adaptive Responses

Nitric oxide (.NO) regulates mitochondrial oxygen uptake through reversible high-affinity binding to heme and Cu2+ centers of cytochrome oxidase, and sets adaptive responses to changes in blood flow, O2 availability and hypoxia, thyroid disorders, endotoxemia and cold adaptation. Moreover, subcellular traffic of.NO synthases (NOS) participates in the adjustment of mechanical cardiac efficiency. In isolated beating rat hearts,.NO and bradykinin promote a dosedependent decrease of myocardial O2 uptake and coronary perfusion pressure, without significant variations of the developed left ventricular pressure. Also, in human hearts.NO from NOS isoforms tunes mechanical performance. On these bases, NOS traffic to mitochondria acquires physiological significance. In liver and heart, mitochondrial NOS (mtNOS) was identified as the neuronal isoform (nNOSα) with postranslational modifications. This new class of NOS spatial confinement leads cells to save energy under particular circumstances. Likewise, thyroid status reciprocally regulates mtNOS expression and.NO yield, while it linearly regulates mitochondrial respiration and systemic oxygen uptake. In rats exposed to cold, a clear contribution of.NO from mtNOS to modulation of.VO2 and redistribution of energy expenditure was reported. We conclude that.NOS traffic and mtNOS activity are natural mechanisms to link mitochondria and cardiovascular responses to environmental and endogenously mediated challenges Keywords: Modulation of oxygen uptake, oxygen delivery, hypoxia, mechanoenergetic coupling, thyroid status, and cold acclimation

Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart

Heart contraction is characterized by the absence of changes in energetic intermediates in response to a large increase of activity. Until now no experimental approach could address this question concerning the intact beating heart. Ca(2+) plays a crucial role in the excitation-contraction coupling, and in vitro studies have evidenced that Ca(2+) may also directly activate mitochondrial oxidative phosphorylation. We applied our new in situ modular control and regulation analysis on isolated beating rat heart perfused under two different calcium concentrations with pyruvate or glucose as the substrate. Modular control analysis demonstrated experimentally that, although control by energy production was slightly higher under glucose conditions compared with pyruvate, most of the control of heart contraction resides in energy utilization. This behavior is the direct consequence of the high sensitivity (elasticity) of the energy producer processes to ATP utilization. Interestingly, the increase in heart metabolic rate by Ca(2+) did not significantly change the pattern of control distribution. The regulation analysis performed under the two calcium conditions demonstrated a balanced activation of myofibrils ATPases, and mitochondrial ATP synthesis in response to Ca(2+) increase. This first study demonstrates in situ the hypothesis that the energetic adequation in heart contraction is mediated by a parallel activation of both processes of energy production and utilization by Ca(2+). The results presented here show that modular control and regulation analyses allow in situ study of internal regulations in intact beating heart energetics and function and may now be applied to heart dysfunctions and therapeutic effects.

Proteome Analysis of Isolated Perfused Organ Effluent as a Novel Model for Protein Biomarker Discovery

The discovery of novel serological biomarkers is critical for improving disease diagnosis and monitoring treatment response. Proteomic analysis of model systems, such as isolated cells in culture and patient plasma and serum, represents the current state-of-the-art. Here, we coupled proteomics with isolated organ perfusion, which allows a disease state to be studied in a physiologic, yet controlled, environment. Potential markers specific to the disease or to changes in the surrounding tissue may be discovered. The effectiveness of this model was evaluated using proteomic analysis of effluent fractions collected from isolated beating rat hearts during reperfusion after brief episodes of ischemia. The detection of clinical markers for myocardial ischemia in this effluent was robust and analytically straightforward, validating the potential of isolated organ perfusion in diagnostic protein discovery.

Ratiometric intracellular calcium imaging in the isolated beating rat heart using indo-1 fluorescence.

Abnormalities in intracellular calcium (Ca(i)(2+)) handling have been implicated as the underlying mechanism in a large number of pathologies in the heart. Study into the relation between Ca(i)(2+) behavior and performance of the whole heart function could provide detailed information into the cellular basis of heart function. In this study we describe an optical ratio imaging setup and an analysis method for the beat-to-beat Ca(i)(2+) videofluorescence images of an indo-1 loaded, isolated Tyrode-perfused beating rat heart. The signal-to-noise ratio and the spatiotemporal resolution (with an optimum of 1 ms and 0.6 mm, respectively) made it possible to register different temporal Ca(i)(2+) transients together with left ventricle pressure changes. The Ca(i)(2+) transients showed that Ca(i)(2+) activation propagates horizontally from left to right during sinus rhythm or from the stimulus site during direct left ventricle stimulation. The indo-1 ratiometric video technique developed allows the imaging of ratio changes of Ca(i)(2+) with a high temporal (1 ms) and spatial (0.6 mm) resolution in the isolated Tyrode-perfused beating rat heart.

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.

NMR-microscopy with TrueFISP at 11.75 T

The purpose of this paper is to demonstrate that a fully balanced gradient echo technique (TrueFISP) can be used for microscopic experiments at high static magnetic field strengths. TrueFISP experiments were successfully performed on homogeneous and inhomogeneous objects at 11.75 T. High-resolution TrueFISP images were obtained from phantoms, plants, formalin-fixed samples, and from an isolated beating rat heart with an in-plane resolution of 78 μm and a slice thickness of 500 μm. The signal-to-noise ratio (SNR) gain of TrueFISP compared to conventional gradient echo or spin echo sequences will allow faster acquisition times or an improvement in spatial resolution for microscopic experiments.

Combined High-Speed NMR Imaging of Perfusion and Microscopic Coronary Conductance Vessels in the Isolated Rat Heart

Noninvasive characterization of microcirculation at the level of both coronary conductance and resistance vessels is of major importance for the understanding of microvascular adaptive processes in the heart. The objective of this study was to determine simultaneously myocardial perfusion and microvessel diameters in the myocardium by magnetic resonance (MR) imaging within the same heart. A MR imaging method is presented which combines high-resolution perfusion measurement (140 × 140 μm2) by spin labeling with flow-weighted MR microscopy of coronary microvessels (φ > 140 μm). We determined changes in myocardial perfusion and vessel diameters of isolated beating rat hearts (n = 10) at rest and during administration of nitroglycerin (0.5 mg/min). Alterations in perfusion were validated by microsphere measurements. Under the influence of nitroglycerin an increase in perfusion (+2.51 ± 0.4 ml · min−1 · g−1, mean ± SEM) and vessel diameters (+14.22 ± 1.92%) could be observed. Endocardial perfusion revealed a modest enhanced susceptibility to nitroglycerin in comparison to epicardial perfusion. Analysis of vessels according to their diameters showed no significant differences. MR imaging allows the noninvasive and simultaneous determination of conducting arteries and smaller resistance vessels in one and the same beating rat heart. Due to an excellent spatial resolution of these methods, transmural characterization of both parameters at rest and during vasodilation is feasible.

Effects of sustained low-flow ischemia and reperfusion on Ca2+ transients and contractility in perfused rat hearts.

We investigated changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and in left ventricular contractility during sustained ischemia and reperfusion in isolated beating rat hearts. Hearts from male Sprague-Dawley rats were perfused retrogradely and were loaded with 4 microM fura-2. Low-flow global ischemia was induced by reducing perfusion flow to 10% and by electric pacing. The hearts were exposed to ischemia for 10 min or 30 min and then reperfused. [Ca2+]i was measured by monitoring the ratio of 500 nm fluorescence excited at 340 and 380 nm while simultaneously measuring left ventricular pressure (LVP). To determine diastolic [Ca2+]i, background autofluorescence was subtracted. LVP rapidly decreased from 82.3+/-8.2 to 17.1+/-2.9 mmHg , whereas the amplitude of the Ca2+ transient did not change significantly during the first 1 min of ischemia. After 10 min of ischemia, the amplitude decreased to 60.8+/-10.6% (p < 0.05) and diastolic [Ca2+]i increased by 26.3+/-2.9% (p < 0.001) compared with the pre-ischemic value (n = 8). When the hearts were reperfused after 10 min of ischemia, the amplitude of the Ca2+ transient and LVP recovered to 79.0+/-7.2% and 73.2+/-7.5 mmHg, respectively. Whereas diastolic [Ca2+]i decreased to the preischemic value. In the hearts exposed to 30 min of ischemia (n = 10), diastolic [Ca2+]i increased even further by 32.7+/-5.3% at the end of ischemia and continued increasing during the 10 min of reperfusion by 42.6+/-15.6%. Six of 10 hearts developed ventricular fibrillation (VF) and intracellular Ca2+ overload after reperfusion. Recovery of LVP after reperfusion was significantly smaller in the hearts exposed to 30 min of ischemia than in the hearts exposed to 10 min of ischemia (58.9+/-11.7 vs. 97.2+/-3.0% of pre-ischemic value, p < 0.05). Diastolic [Ca2+]i also increased under hypoxic conditions (N2 bubbling) in this model. These results suggest that increases in diastolic [Ca2+]i might play an important role in myocardial contractile dysfunction and viability in ischemia-reperfusion injury.

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart.

Isolated rat heart perfused with 1.5-7.5 microM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2 uptake from 3.2 +/- 0.3 to 1.6 +/- 0.1 (7.5 microM NO, n = 18, P < 0.05) and to 1.2 +/- 0.1 microM O2.min-1.g tissue-1 (10 microM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with an induced release of hydrogen peroxide (H2O2) in the effluent (r = 0.99, P < 0.01). NO markedly decreased the O2 uptake of isolated rat heart mitochondria (50% inhibition at 0.4 microM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and cytochrome c, which accounts for the effects in O2 uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1-0.3 microM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2 uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.

Study of Microcirculation by Coloured Microspheres and NMR-microscopy in Isolated Rat Heart: Effect of Ischaemia, Endothelin-1 and Endothelin-1 Antagonist BQ 610

Although the investigation of coronary microcirculation is of great importance, available methods have severe restrictions. They do not allow the study of vasodynamics of resistance vessels and microscopic conductance vessels simultaneously in the isolated beating rat heart. We now demonstrate that the combined measurement of perfusion which reflects the state of resistance vessels and cross-sections of microscopic conductance vessels is feasible in the model of the isolated constant flow perfused rat heart. Perfusion measurement was based on injection of coloured microspheres. Cross-sections of microscopic conductance vessels (diameter >140μm) were determined by NMR-microscopy by flow weighted imaging. Both methods were established recently by our group. The combined measurement was applied to hearts which were subjected to ischaemia and reperfusion (group 1:n=5, 15 min ischaemia/group 2:n=7, 30 min ischaemia/measurements before ischaemia and 15/30 min after reperfusion), 200 pmol endothelin-1 bolus application (group 3:n=6/measurements before and 5 min after drug application), continuous infusion of the endothelin-1 antagonist BQ 610 (group 4:n=6/measurements before and 20 min after onset of infusion), and 200 pmol endothelin-1 application superimposed on 20 min of continuous BQ 610 infusion (group 5:n=7/combined measurement before BQ 610 infusion and 5 min after endothelin-1 application). In group 1, 15 min reperfusion restored the pre-ischaemic perfusion state, whereas conductance vessels were dilated (80.8±2.6%), after 30 min reperfusion pre-ischaemic conditions were also restored for conductance vessels. In group 2, a redistribution of perfusion from left ventricular endocardium to the right ventricular wall was observed. Post-ischaemic rhythm disturbances made NMR-imaging in this group impossible. In group 3, a shift of perfusion from the left ventricular myocardium to the right ventricular wall was observed. Similarly, the cross-section of left ventricular conductance vessels decreased (−32.6±2.1%), whereas size of right ventricular vessels increased. In group 4, BQ 610 had no effect on perfusion nor on vessel size and antagonized the effect of endothelin-1 on perfusion and vessel size in group 5.

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 image enabled visualization of the time-dependent alterations in the free radical distribution and anatomical structure of the heart that occur during the cardiac cycle.

Real-time volume measurements in the isolated beating rat heart

A new technique, utilizing scattered X-rays, has been developed that is capable of measuring isolated working rat heart left ventricular (LV) volumes continuously throughout the cardiac cycle. The technique was validated by comparing cardiac outputs measured via fluid collections (r = 0.959, n = 10). The new methodology will permit real-time measurements of pressure-volume relationships, stroke work, and indexes of diastolic function in the isolated working rat heart.

Protective Effects of Calcium Antagonists on Energy and Substrate Metabolism During Ischemia and Reperfusion in Hypertensive Myocardial Hypertrophy

The aim of the present study was to define the protective effects of verapamil and nifedipine on mechanical performance and energy and substrate metabolism of the postischemically reperfused myocardium in a chronic pressure overload cardiac hypertrophy model. The isolated beating rat heart preparation was used and left ventricular pressures and high-energy phosphates were continuously monitored during 30 min of global ischemia and reperfusion, respectively. Recovery of mechanical performance and high-energy phosphate and sugar monophosphate metabolism was significantly impaired in untreated hypertrophied hearts compared with normal control hearts and hypertrophied hearts treated with calcium antagonists. In hypertrophied hearts with chronic verapamil treatment, recovery was significantly improved compared to acute verapamil treatment, nifedipine treatment, and normal control hearts. Thus, verapamil and nifedipine exerted a protective effect on the postischemic recovery of the hypertrophied myocardium that was most prominent following long-term pretreatment with verapamil. Prolonged maintenance of adequate plasma levels and tissue distribution of calcium antagonists before an ischemic event may improve the postischemic prognosis in the presence of pressure-induced myocardial hypertrophy.

Transcapillary Permeability and Subendothelial Distribution of Endothelial and Amniotic Fluid Insulin- Like Growth Factor Binding Proteins in the Rat Heart*

Insulin-like growth factor (IGF) binding proteins (IGFBP) were purified from conditioned media of cultured bovine endothelial cells (ECBP) and from human amniotic fluid (IGFBP-1), and then labeled by radioiodination. 125I-ECBP and 125I-IGFBP-1 were perfused through isolated beating rat hearts for 1 and 5 min, and the hearts fixed and analyzed for 125I-BP content and distribution. One to 4% of the perfused 125I-ECBP and 125I-IGFBP-1 crossed the capillary boundary. The ECBPs predominantly localized as intact 125I-BP in connective tissue elements of the heart with less 125I-BP in cardiac muscle. The ratio of 125I-ECBP in connective tissue: muscle (normalized to percent vol of these compartments) was greater than or equal to 10:1. In contrast, the IGFBP-1 had a greater affinity for cardiac muscle with ratios of 125I-IGFBP-1 in connective tissue:muscle of approximately 1:2. When 125I-IGF-I, in the absence of any BPs, was perfused through the hearts approximately 3-5% left the microcirculation and was found in subendothelial tissues. 125I-IGF-I localized primarily to cardiac muscle with a distribution of connective tissue:cardiac muscle of about 1:3. The findings in the isolated perfused heart were confirmed in intact animals. After 125I-IGFBP-1 was injected into anesthetized rats and allowed to circulate for 5 min, substantial radioactivity was associated with the heart. As in the isolated heart, the IGFBP-1 preferentially localized to cardiac muscle with a connective tissue:cardiac muscle ratio of 1:3. We conclude that IGFBPs produced by endothelial cells and the IGFBP-1 contained in amniotic fluid can cross the capillary boundaries of the rat heart, and that the ECBPs preferentially localize in connective tissue elements of the myocardium, whereas IGFBP-1 predominantly localizes in cardiac muscle.