Johannes H. G. M. van Beek

A vision and strategy for the virtual physiological human in 2010 and beyond

European funding under framework 7 (FP7) for the virtual physiological human (VPH) project has been in place now for nearly 2 years. The VPH network of excellence (NoE) is helping in the development of common standards, open-source software, freely accessible data and model repositories, and various training and dissemination activities for the project. It is also helping to coordinate the many clinically targeted projects that have been funded under the FP7 calls. An initial vision for the VPH was defined by framework 6 strategy for a European physiome (STEP) project in 2006. It is now time to assess the accomplishments of the last 2 years and update the STEP vision for the VPH. We consider the biomedical science, healthcare and information and communications technology challenges facing the project and we propose the VPH Institute as a means of sustaining the vision of VPH beyond the time frame of the NoE.

Visualization of mitochondria in cardiomyocytes

The simultaneous detection of third harmonic (THG), and multiphoton excitation fluorescence (MPF) or second harmonic (SHG) from the same focal volume has led us to the development of a nonlinear multimodal microscopic biological imaging tool. The multimodal microscope has been applied for imaging of isolated live cardiomyocytes, and investigation of structural origin of the THG and SHG signals has been performed. By employing the different image contrast mechanisms, differentiation of structures inside a single live adult rat cardiomyocyte has been achieved. Based on structural crosscorrelation image analysis between NAD(P)H fluorescence and THG, and morphology of cardiomyocytes we were able to assign large part of the structure revealed by THG to the mitochondria. The crosscorrelation of THG with fluorescence of tetramethylrhodamine methyl ester (TMRM) labeled cardiomyocytes confirmed the mitochondrial origin of THG. The SHG generated structures were anticorrelated with THG and possessed the characteristic pattern of the myofibrils in the myocyte in accordance with the literature. Possible visualization of mitochondria with THG microscopy appeared due to enhancement of the third harmonic by multilayer arrangement of cristae.

A vision and strategy for the virtual physiological human: 2012 update.

European funding under Framework 7 (FP7) for the virtual physiological human (VPH) project has been in place now for 5 years. The VPH Network of Excellence (NoE) has been set up to help develop common standards, open source software, freely accessible data and model repositories, and various training and dissemination activities for the project. It is also working to coordinate the many clinically targeted projects that have been funded under the FP7 calls. An initial vision for the VPH was defined by the FP6 STEP project in 2006. In 2010, we wrote an assessment of the accomplishments of the first two years of the VPH in which we considered the biomedical science, healthcare and information and communications technology challenges facing the project (Hunter et al. 2010 Phil. Trans. R. Soc. A 368, 2595–2614 (doi:10.1098/rsta.2010.0048)). We proposed that a not-for-profit professional umbrella organization, the VPH Institute, should be established as a means of sustaining the VPH vision beyond the time-frame of the NoE. Here, we update and extend this assessment and in particular address the following issues raised in response to Hunter et al.: (i) a vision for the VPH updated in the light of progress made so far, (ii) biomedical science and healthcare challenges that the VPH initiative can address while also providing innovation opportunities for the European industry, and (iii) external changes needed in regulatory policy and business models to realize the full potential that the VPH has to offer to industry, clinics and society generally.

CK inhibition accelerates transcytosolic energy signaling during rapid workload steps in isolated rabbit hearts

The effect of graded creatine kinase (CK) inhibition on the response time of mitochondrial O2 consumption to dynamic workload jumps (tmito) was studied in isolated rabbit hearts. Tyrode-perfused hearts (n = 7/group) were exposed to 15 min of 0, 0.1, 0.2, or 0.4 mM iodoacetamide (IA) (CK activity = 100, 14, 6, and 3%, respectively). Pretreatment tmito was similar across groups at 6.5 +/- 0.5 s (mean +/- SE). The increase observed over time in control hearts (33 +/- 8%) was progressively reversed to 16 +/- 6, -20 +/- 6 (P < 0.01 vs. control), and -46 +/- 6 (P < 0.01 vs. control) % in the 0.1, 0.2 and 0.4 mM IA groups, respectively. The faster response times occurred without reductions in mitochondrial oxidative capacity (assessed in vitro) or myocardial O2 consumption of the whole heart during workload steps. Isovolumic contractile function assessed as rate-pressure product (RPP) and contractile reserve (increase in RPP during heart rate steps) were significantly reduced by IA. We conclude that CK in the myofibrils and/or cytosol does not speed up transfer of the energy-related signal to the mitochondria but rather acts as an energetic buffer, effectively slowing the stimulus between myofibrils/ion pumps and oxidative phosphorylation. This argues against the existence of an obligatory creatine phosphate energy shuttle, because CK is effectively bypassed.The effect of graded creatine kinase (CK) inhibition on the response time of mitochondrial O2 consumption to dynamic workload jumps ( t mito) was studied in isolated rabbit hearts. Tyrode-perfused hearts ( n = 7/group) were exposed to 15 min of 0, 0.1, 0.2, or 0.4 mM iodoacetamide (IA) (CK activity = 100, 14, 6, and 3%, respectively). Pretreatment t mito was similar across groups at 6.5 ± 0.5 s (mean ± SE). The increase observed over time in control hearts (33 ± 8%) was progressively reversed to 16 ± 6, -20 ± 6 ( P< 0.01 vs. control), and -46 ± 6 ( P < 0.01 vs. control) % in the 0.1, 0.2 and 0.4 mM IA groups, respectively. The faster response times occurred without reductions in mitochondrial oxidative capacity (assessed in vitro) or myocardial O2 consumption of the whole heart during workload steps. Isovolumic contractile function assessed as rate-pressure product (RPP) and contractile reserve (increase in RPP during heart rate steps) were significantly reduced by IA. We conclude that CK in the myofibrils and/or cytosol does not speed up transfer of the energy-related signal to the mitochondria but rather acts as an energetic buffer, effectively slowing the stimulus between myofibrils/ion pumps and oxidative phosphorylation. This argues against the existence of an obligatory creatine phosphate energy shuttle, because CK is effectively bypassed.

Assessing heterogeneous distribution of blood flow and metabolism in the heart

Abstract The literature is reviewed on methods to assess heterogeneity of blood flow, substrate uptake and oxidative end energy metabolism in the normal heart, and their interrelations. Even though the factors controlling matching on the regional level remain largely obscure, the evidence that heterogeneous blood flow partially correlates to indicators of metabolism in the normal heart is accumulating, particularly in face of a correlation between acetate metabolism indicative of regional O2 consumption to microsphere blood flow. Moreover, the partial matching cannot be explained by vascular anatomical differences from one region to the other, since, although fractal theory can partially describe the branching patterns of the coronaries, vasodilation is similar among regions upon metabolic stimulation of the heart. It is dissimilar among regions, so that blood flow is redistributed, upon maximum vasodilation with adenosine or hypoxia, denoting regionally different maximum vessel diameter and flow reserve. However, regionally differing tissue composition could also contribute somewhat to regional differences in (the need for) blood flow. It is still unknown, because of technical limitations, how the foregoing measures relate to regional work load.

Analyzing the Functional Properties of the Creatine Kinase System with Multiscale ‘Sloppy’ Modeling

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The ‘phosphocreatine shuttle’ hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using ‘sloppy modeling’ ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 µM*s−1) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 µM*s−1, while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 µM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect.

Measurement of the Oxygenation Status of the Isolated Perfused Rat Heart Using Near Infrared Detection

The oxygenation status of isolated hearts has often been measured using optical detection methods for the visible range of the spectrum (Tamura et al., 1989). To the best of our knowledge there are no reports on measurement of myoglobin oxygenation using detection methods in the near-infrared region of the spectrum. On the other hand, equipment for near-infrared measurements on tissue is widely available and is in some instances quite inexpensive. Therefore we investigated in the present study whether near-infrared (NIR) detection at two wavelengths provides a way to conveniently determine myoglobin oxygenation in an isolated organ as small as the saline-perfused rat heart.

Multiscale and modular analysis of cardiac energy metabolism: repairing the broken interfaces of isolated system components.

Computational models of large molecular systems can be assembled from modules representing biological function emerging from interactions among a small subset of molecules. Experimental information on isolated molecules can be integrated with the response of the network as a whole to estimate crucial missing parameters. As an example, a “skeleton” model is analyzed for the module regulating dynamic adaptation of myocardial oxidative phosphorylation (OxPhos) to fluctuating cardiac energy demand. The module contains adenine nucleotides, creatine, and phosphate groups. Enzyme kinetic equations for two creatine kinase (CK) isoforms were combined with the response time of OxPhos (tmito; generalized time constant) to steps in the cardiac pacing rate to identify all module parameters. To obtain tmito, the time course of O2 uptake was measured for the whole heart. An O2 transport model was used to deconvolute the whole‐heart response to the mitochondrial level. By optimizing mitochondrial outer membrane permeability to 21 μm/s the experimental tmito = 3.7 s was reproduced. This in vivo value is about four times larger, or smaller, respectively, than conflicting values obtained from two different in vitro studies. This demonstrates an important rule for multiscale analysis: experimental responses and modeling of the system at the larger scale allow one to estimate essential parameters for the interfaces of components which may have been altered during physical isolation. The model correctly predicts a smaller tmito when CK activity is reduced. The model further predicts a slower response if the muscle CK isoform is overexpressed and a faster response if mitochondrial CK is overexpressed. The CK system is very effective in decreasing maximum levels of ADP during systole and reducing average Pi levels over the whole cardiac cycle.

Dynamics of tissue oxygenation in isolated rabbit heart as measured with near-infrared spectroscopy

We investigated the role of myoglobin (Mb) in supplying O2 to mitochondria during transitions in cardiac workload. Isovolumic rabbit hearts ( n = 7) were perfused retrogradely with hemoglobin-free Tyrode solution at 37°C. Coronary venous O2 tension was measured polarographically, and tissue oxygenation was measured with two-wavelength near-infrared spectroscopy (NIRS), both at a time resolution of ∼2 s. During transitions to anoxia, 68 ± 2% (SE) of the NIRS signal was due to Mb and the rest to cytochrome oxidase. For heart rate steps from 120 to 190 or 220 beats/min, the NIRS signal decreased significantly by 6.9 ± 1.3 or 11.1 ± 2.1% of the full scale, respectively, with response times of 11.0 ± 0.8 or 9.1 ± 0.5 s, respectively. The response time of end-capillary O2 concentration ([O2]), estimated from the venous [O2], was 8.6 ± 0.8 s for 190 beats/min ( P< 0.05 vs. NIRS time) or 8.5 ± 0.9 s for 220 beats/min ( P > 0.05). The mean response times of mitochondrial O2 consumption (V˙o 2) were 3.7 ± 0.7 and 3.6 ± 0.6 s, respectively. The deoxygenation of oxymyoglobin (MbO2) accounted for only 12-13% of the total decrease in tissue O2, with the rest being physically dissolved O2. During 11% reductions in perfusion flow at 220 beats/min, Mb was 1.5 ± 0.4% deoxygenated ( P < 0.05), despite the high venous [Formula: see text] of 377 ± 17 mmHg, indicating metabolism-perfusion mismatch. We conclude that the contribution of MbO2 to the increase of V˙o 2 during heart rate steps in saline-perfused hearts was small and slow compared with that of physically dissolved O2.

The mitochondrial outer membrane is not a major diffusion barrier for ADP in mouse heart skinned fibre bundles.

The response of mitochondrial oxygen consumption to ADP in saponin-skinned cardiac fibre bundles has an apparent Km an order of magnitude higher than that in isolated mitochondria. Here we report that incubating skinned cardiac fibre bundles from wild-type mice or double-knockout mice lacking both cytosolic and mitochondrial creatine kinase (CK) with CK and creatine or with yeast hexokinase and glucose as extramitochondrial ADP-producing systems decreases the apparent Km of the bundles for ADP severalfold. We conclude that the affinity of mitochondria for ADP in mouse heart is of the same order of magnitude as that of isolated mitochondria, while the high apparent Km of the bundles is caused by diffusion gradients outside the mitochondria.