Pepijn van Horssen

Disruption of Hexokinase II–Mitochondrial Binding Blocks Ischemic Preconditioning and Causes Rapid Cardiac Necrosis

Rationale: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. Objective: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. Methods and Results: Ex vivo perfused HKII+/− hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 &mgr;mol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. Conclusions: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Mitochondrial oxygen tension within the heart

By using a newly developed optical technique which enables non-invasive measurement of mitochondrial oxygenation (mitoPO(2)) in the intact heart, we addressed three long-standing oxygenation questions in cardiac physiology: 1) what is mitoPO(2) within the in vivo heart?, 2) is mitoPO(2) heterogeneously distributed?, and 3) how does mitoPO(2) of the isolated Langendorff-perfused heart compare with that in the in vivo working heart? Following calibration and validation studies of the optical technique in isolated cardiomyocytes, mitochondria and intact hearts, we show that in the in vivo condition mean mitoPO(2) was 35+/-5 mm Hg. The mitoPO(2) was highly heterogeneous, with the largest fraction (26%) of mitochondria having a mitoPO(2) between 10 and 20 mm Hg, and 10% between 0 and 10 mm Hg. Hypoxic ventilation (10% oxygen) increased the fraction of mitochondria in the 0-10 mm Hg range to 45%, whereas hyperoxic ventilation (100% oxygen) had no major effect on mitoPO(2). For Langendorff-perfused rat hearts, mean mitoPO(2) was 29+/-5 mm Hg with the largest fraction of mitochondria (30%) having a mitoPO(2) between 0 and 10 mm Hg. Only in the maximally vasodilated condition, did the isolated heart compare with the in vivo heart (11% of mitochondria between 0 and 10 mm Hg). These data indicate 1) that the mean oxygen tension at the level of the mitochondria within the heart in vivo is higher than generally considered, 2) that mitoPO(2) is considerably heterogeneous, and 3) that mitoPO(2) of the classic buffer-perfused Langendorff heart is shifted to lower values as compared to the in vivo heart.

Coronary structure and perfusion in health and disease

Blood flow is distributed through the heart muscle via a system of vessels forming the coronary circulation. The perfusion of the myocardium can be hampered by atherosclerosis creating localized obstructions in the epicardial vessels or by microvascular disease. In early stages of the disease, these impediments to blood flow are offset by dilation of the resistance vessels, which normally compensates for a decrease in perfusion pressure or increased metabolism. However, this dilatory reserve can become exhausted, which in general occurs first at the deeper layers of the heart wall where intramural vessels are subjected to compressive forces related to heart contraction. In the catheterization laboratory, guide wires of 0.33 mm diameter are available that are equipped with a pressure and flow velocity sensor at the tip, which can be positioned distal to the stenosis. These signals provide information about the impediment of the stenosis on coronary flow and allow for the evaluation of the status of the microcirculation. However, the interpretation of these signals is strongly model-dependent and therefore it is of paramount importance to develop realistic models reflecting the anatomy and unique physiology of the coronary circulation.

3D Imaging of vascular networks for biophysical modeling of perfusion distribution within the heart

One of the main determinants of perfusion distribution within an organ is the structure of its vascular network. Past studies were based on angiography or corrosion casting and lacked quantitative three dimensional, 3D, representation. Based on branching rules and other properties derived from such imaging, 3D vascular tree models were generated which were rather useful for generating and testing hypotheses on perfusion distribution in organs. Progress in advanced computational models for prediction of perfusion distribution has raised the need for more realistic representations of vascular trees with higher resolution. This paper presents an overview of the different methods developed over time for imaging and modeling the structure of vascular networks and perfusion distribution, with a focus on the heart. The strengths and limitations of these different techniques are discussed. Episcopic fluorescent imaging using a cryomicrotome is presently being developed in different laboratories. This technique is discussed in more detail, since it provides high-resolution 3D structural information that is important for the development and validation of biophysical models but also for studying the adaptations of vascular networks to diseases. An added advantage of this method being is the ability to measure local tissue perfusion. Clinically, indices for patient-specific coronary stenosis evaluation derived from vascular networks have been proposed and high-resolution noninvasive methods for perfusion distribution are in development. All these techniques depend on a proper representation of the relevant vascular network structures.

Porcine coronary collateral formation in the absence of a pressure gradient remote of the ischemic border zone

In the current paradigm on coronary collateral development, it is assumed that these vessels develop consequentially from increased fluid shear stress (FSS) through preexisting collateral arteries. The increased FSS follows from an increase in pressure gradient between the region at risk and well-perfused surroundings. The objective of this study was to test the hypothesis that, in the heart, collateral connections can form in the absence of an increased FFS and consequentially at any depth and region within the ventricular wall. In Yorkshire pigs, gradual left circumflex coronary artery occlusion was obtained over 6 wk by an ameroid constrictor, whereas the control group underwent a sham operation. Hearts were harvested and subsequently processed in an imaging cryomicrotome, resulting in 40-μm voxel resolution three-dimensional reconstructions of the intramural vascular vessels. Dedicated software segmented the intramural vessels and all continuous vascular pathways containing a collateral connection. In the ameroid group, 192 collaterals, 22-1,049 μm in diameter, were detected with 62% within the subendocardium. Sixty percent of collaterals bridged from the left anterior descending artery to left circumflex coronary artery. A novel result is that 25% (n = 48) of smaller-radius collaterals (P = 0.047) connected with both origin and terminus in the nontarget area where perfusion was assumed uncompromised. In the porcine heart, collateral vessels develop not only in ischemic border zones with increased FSS but also away from such border zones where increased FSS is unlikely. The majority of collaterals were located at the subendocardium, corresponding to the region with highest prevalence for ischemia.

Stimulation of Coronary Collateral Growth by Granulocyte Stimulating Factor. Role of Reactive Oxygen Species

Objective—The purpose of this study was to determine whether G-CSF promotes coronary collateral growth (CCG) and decipher the mechanism for this stimulation. Methods and Results—In a rat model of repetitive episodic myocardial ischemia (RI, 40 seconds LAD occlusion every 20 minutes for 2 hours and 20 minutes, 3 times/d for 5 days) CCG was deduced from collateral-dependent flow (flow to LAD region during occlusion). After RI, G-CSF (100 &mgr;g/kg/d) increased CCG (P<0.01) (0.47±0.15) versus vehicle (0.14±0.06). Surprisingly, G-CSF treatment without RI increased CCG (0.57±0.18) equal to G-CSF+RI. We evaluated ROS by dihydroethidine (DHE) fluorescence (LV injection, 60 &mgr;g/kg, during two episodes of ischemia). DHE fluorescence was double in G-CSF+RI versus vehicle+RI (P<0.01), and even higher in G-CSF without RI (P<0.01). Interestingly, the DHE signal did not colocalize with myeloperoxidase (immunostaining, neutrophil marker) but appeared in cardiac myocytes. The study of isolated cardiac myocytes revealed the cytokine stimulates ROS which elicit production of angiogenic factors. Apocynin inhibited G-CSF effects both in vivo and in vitro. Conclusions—G-CSF stimulates ROS production directly in cardiomyocytes, which plays a pivotal role in triggering adaptations of the heart to ischemia including growth of the coronary collaterals.

Immediate Effect of Kidney Cryoablation on Renal Arterial Structure in a Porcine Model Studied by Imaging Cryomicrotome

PURPOSE Injury to blood microvessels has a crucial role in effective cryoablation for renal masses. We visualized vascular injury induced by a clinically applied cryoablation instrument and established a microvascular diameter threshold for vascular damage. MATERIALS AND METHODS In 5 anesthetized pigs 1 kidney each was exposed and 3, 17 gauge cryoneedles were inserted in 1 pole. Tissue was exposed to freezing for 2 x 10 minutes with a 10-minute thaw between freezes. After nephrectomy the arteries were injected with fluorescence dyed casting material and the kidney was frozen to -20C and cut in 40 to 60 micron slices in the imaging cryomicrotome, where fluorescent images of the cutting plane of the bulk were obtained. This resulted in a 3-dimensional image of the arterial tree that was segmented, resulting in unbranched vessel segments. Histograms were constructed with the total segment length per diameter bin plotted as function of diameter. RESULTS The ablated zone was sharply demarcated on fluorescent and normal light images. Mean +/- SD diameter at the peak of the histogram from control areas was 152.4 +/- 5.3 micron. Compared to control areas the peak diameter of ablated areas was shifted to a larger diameter by an average of 25.4 +/- 2.6 micron. CONCLUSIONS Immediate renal cryoablation injury destroys arteries smaller than 180 micron. Branching structures of larger arteries remain anatomically intact and connected to vascular structures in surrounding tissue.

Quantitative assessment of magnetic resonance derived myocardial perfusion measurements using advanced techniques: microsphere validation in an explanted pig heart system

BackgroundCardiovascular Magnetic Resonance (CMR) myocardial perfusion imaging has the potential to evolve into a method allowing full quantification of myocardial blood flow (MBF) in clinical routine. Multiple quantification pathways have been proposed. However at present it remains unclear which algorithm is the most accurate. An isolated perfused, magnetic resonance (MR) compatible pig heart model allows very accurate titration of MBF and in combination with high-resolution assessment of fluorescently-labeled microspheres represents a near optimal platform for validation. We sought to investigate which algorithm is most suited to quantify myocardial perfusion by CMR at 1.5 and 3 Tesla using state of the art CMR perfusion techniques and quantification algorithms.MethodsFirst-pass perfusion CMR was performed in an MR compatible blood perfused pig heart model. We acquired perfusion images at physiological flow (“rest”), reduced flow (“ischaemia”) and during adenosine-induced hyperaemia (“hyperaemia”) as well as during coronary occlusion. Perfusion CMR was performed at 1.5 Tesla (n = 4 animals) and at 3 Tesla (n = 4 animals). Fluorescently-labeled microspheres and externally controlled coronary blood flow served as reference standards for comparison of different quantification strategies, namely Fermi function deconvolution (Fermi), autoregressive moving average modelling (ARMA), exponential basis deconvolution (Exponential) and B-spline basis deconvolution (B-spline).ResultsAll CMR derived MBF estimates significantly correlated with microsphere results. The best correlation was achieved with Fermi function deconvolution both at 1.5 Tesla (r = 0.93, p < 0.001) and at 3 Tesla (r = 0.9, p < 0.001). Fermi correlated significantly better with the microspheres than all other methods at 3 Tesla (p < 0.002). B-spline performed worse than Fermi and Exponential at 1.5 Tesla and showed the weakest correlation to microspheres (r = 0.74, p < 0.001). All other comparisons were not significant. At 3 Tesla exponential deconvolution performed worst (r = 0.49, p < 0.001).ConclusionsCMR derived quantitative blood flow estimates correlate with true myocardial blood flow in a controlled animal model. Amongst the different techniques, Fermi function deconvolution was the most accurate technique at both field strengths. Perfusion CMR based on Fermi function deconvolution may therefore emerge as a useful clinical tool providing accurate quantitative blood flow assessment.

Innate collateral segments are predominantly present in the subendocardium without preferential connectivity within the left ventricular wall

Innate collateral arteries provide the biophysical substrate for arteriogenesis. Their distribution and morphology predestine tissue areas salvageable by collateral flow. Fluorescent episcopic cryomicrotome imaging resulted in a three‐dimensional representation of the coronary network, in which collateral segments were automatically identified. Innate collateral segments are predominantly present in the subendocardium without preferential connectivity within the left ventricular wall of the dog heart and are preferentially oriented perpendicular to the long axis of the heart in the outer layers and parallel to this axis at the subendocardium. These results suggest that collateral segments are maintained without local hypoxia but because of heterogeneity in pressure gradients in the arterial tree. The high density and long‐axis orientation of collateral arteries in the subendocardial region provide the substrate for arterial plexus formation and indicate the need for three‐dimensional perfusion assessment in clinical perfusion imaging.

Myocardial perfusion distribution and coronary arterial pressure and flow signals:clinical relevance in relation to multiscale modeling, a review

Abstract Coronary artery disease, CAD, is associated with both narrowing of the epicardial coronary arteries and microvascular disease, thereby limiting coronary flow and myocardial perfusion. CAD accounts for almost 2 million deaths within the European Union on an annual basis. In this paper, we review the physiological and pathophysiological processes underlying clinical decision making in coronary disease as well as the models for interpretation of the underlying physiological mechanisms. Presently, clinical decision making is based on non-invasive magnetic resonance imaging, MRI, of myocardial perfusion and invasive coronary hemodynamic measurements of coronary pressure and Doppler flow velocity signals obtained during catheterization. Within the euHeart project, several innovations have been developed and applied to improve diagnosis-based understanding of the underlying biophysical processes. Specifically, MRI perfusion data interpretation has been advanced by the gradientogram, a novel graphical representation of the spatiotemporal myocardial perfusion gradient. For hemodynamic data, functional indices of coronary stenosis severity that do not depend on maximal vasodilation are proposed and the Valsalva maneuver for indicating the extravascular resistance component of the coronary circulation has been introduced. Complementary to these advances, model innovation has been directed to the porous elastic model coupled to a one-dimensional model of the epicardial arteries. The importance of model development is related to the integration of information from different modalities, which in isolation often result in conflicting treatment recommendations.