Toward a grey box approach for cardiovascular physiome

The physiomic approach is now widely used in the diagnosis of cardiovascular diseases. There are two possible methods for cardiovascular physiome: the traditional mathematical model and the machine learning (ML) algorithm. ML is used in almost every area of society for various tasks formerly performed by humans. Specifically, various ML techniques in cardiovascular medicine are being developed and improved at unprecedented speed. The benefits of using ML for various tasks is that the inner working mechanism of the system does not need to be known, which can prove convenient in situations where determining the inner workings of the system can be difficult. The computation speed is also often higher than that of the traditional mathematical models. The limitations with ML are that it inherently leads to an approximation, and special care must be taken in cases where a high accuracy is required. Traditional mathematical models are, however, constructed based on underlying laws either proven or assumed. The results from the mathematical models are accurate as long as the model is. Combining the advantages of both the mathematical models and ML would increase both the accuracy and efficiency of the simulation for many problems. In this review, examples of cardiovascular physiome where approaches of mathematical modeling and ML can be combined are introduced.

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca2+]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca2+], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca2+] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca2+], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca2+] and TMRE for Psi(m) or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca2+] concentration was 1.03 microM. This 1 microM cytosolic Ca2+ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca2+] increase was limited to ~30 microM in the presence of 1 microM cytosolic Ca2+. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

Potassium Currents in Isolated Deiters' Cells of Guinea Pig

Deiters' cells are the supporting cells in organ of Corti and are suggested to play an important role in biochemical and mechanical modulation of outer hair cells. We successfully isolated functionally different K+ currents from Deiters' cells of guinea pig using whole cell patch clamp technique. With high K+ pipette solution, depolarizing step pulses activated strongly outward rectifying currents which were dose-dependently blocked by clofilium, a class III anti-arrhythmic K+ channel blocker. The remaining outward current was transient in time course whereas the clofilium-sensitive outward current showed slow inactivation and delayed rectification. Addition of 5 mM tetraethylammonium (TEA) further blocked the remaining current leaving a very fast inactivating transient outward current. Therefore, at least three different types of K+ current were identified in Deiters' cells, such as fast activating and fast inactivating current, fast activating slow inactivating current, and very fast inactivating transient outward current. Physiological role of them needs to be established.

A Computational Model of Cytosolic and Mitochondrial Ca2+ in Paced Rat Ventricular Myocytes

We carried out a series of experiment demonstrating the role of mitochondria in the cytosolic and mitochondrial Ca2+ transients and compared the results with those from computer simulation. In rat ventricular myocytes, increasing the rate of stimulation (1~3 Hz) made both the diastolic and systolic [Ca2+] bigger in mitochondria as well as in cytosol. As L-type Ca2+ channel has key influence on the amplitude of Ca2+-induced Ca2+ release, the relation between stimulus frequency and the amplitude of Ca2+ transients was examined under the low density (1/10 of control) of L-type Ca2+ channel in model simulation, where the relation was reversed. In experiment, block of Ca2+ uniporter on mitochondrial inner membrane significantly reduced the amplitude of mitochondrial Ca2+ transients, while it failed to affect the cytosolic Ca2+ transients. In computer simulation, the amplitude of cytosolic Ca2+ transients was not affected by removal of Ca2+ uniporter. The application of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) known as a protonophore on mitochondrial membrane to rat ventricular myocytes gradually increased the diastolic [Ca2+] in cytosol and eventually abolished the Ca2+ transients, which was similarly reproduced in computer simulation. The model study suggests that the relative contribution of L-type Ca2+ channel to total transsarcolemmal Ca2+ flux could determine whether the cytosolic Ca2+ transients become bigger or smaller with higher stimulus frequency. The present study also suggests that cytosolic Ca2+ affects mitochondrial Ca2+ in a beat-to-beat manner, however, removal of Ca2+ influx mechanism into mitochondria does not affect the amplitude of cytosolic Ca2+ transients.

Modeling of Arrhythmogenic Automaticity Induced by Stretch in Rat Atrial Myocytes

Since first discovered in chick skeletal muscles, stretch-activated channels (SACs) have been proposed as a probable mechano-transducer of the mechanical stimulus at the cellular level. Channel properties have been studied in both the single-channel and the whole-cell level. There is growing evidence to indicate that major stretch-induced changes in electrical activity are mediated by activation of these channels. We aimed to investigate the mechanism of stretch-induced automaticity by exploiting a recent mathematical model of rat atrial myocytes which had been established to reproduce cellular activities such as the action potential, Ca2+ transients, and contractile force. The incorporation of SACs into the mathematical model, based on experimental results, successfully reproduced the repetitive firing of spontaneous action potentials by stretch. The induced automaticity was composed of two phases. The early phase was driven by increased background conductance of voltage-gated Na+ channel, whereas the later phase was driven by the reverse-mode operation of Na+/Ca2+ exchange current secondary to the accumulation of Na+ and Ca2+ through SACs. These results of simulation successfully demonstrate how the SACs can induce automaticity in a single atrial myocyte which may act as a focus to initiate and maintain atrial fibrillation in concert with other arrhythmogenic changes in the heart.

Implication of phosphorylation of the myosin II regulatory light chain in insulin-stimulated GLUT4 translocation in 3T3-F442A adipocytes

In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca2+/ calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca2+ in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3- F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca2+-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin- stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca2+/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.

Role of Stretch-Activated Channels in Stretch-Induced Changes of Electrical Activity in Rat Atrial Myocytes

We developed a cardiac cell model to explain the phenomenon of mechano-electric feedback (MEF), based on the experimental data with rat atrial myocytes. It incorporated the activity of ion channels, pumps, exchangers, and changes of intracellular ion concentration. Changes in membrane excitability and Ca2+ transients could then be calculated. In the model, the major ion channels responsible for the stretch-induced changes in electrical activity were the stretch-activated channels (SACs). The relationship between the extent of stretch and activation of SACs was formulated based on the experimental findings. Then, the effects of mechanical stretch on the electrical activity were reproduced. The shape of the action potential (AP) was significantly changed by stretch in the model simulation. The duration was decreased at initial fast phase of repolarization (AP duration at 20% repolarization level from 3.7 to 2.5 ms) and increased at late slow phase of repolarization (AP duration at 90% repolarization level from 62 to 178 ms). The resting potential was depolarized from -75 to -61 mV. This mathematical model of SACs may quantitatively predict changes in cardiomyocytes by mechanical stretch.

The Effects of Neurotransmitters on the Ion Channels in the Isolated Deiters'Cells of Guinea Pig Cochlea / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The supporting cells in the organ of Corti help to maintain the structural integrity of the organ, but it has been suggested that they also actively participate in regulating sound transduction. The existence of neural control was implied by the finding of efferent synapses in Deiters' cells, and the fact that the intracellular Ca2+ concentration was increased by the application of neurotransmitters, such as ATP (adenosine triphosphate) and Ach (acetylcholine), resulting in movement of the phalangeal processes of the Deiters' cells. This study investigated the effects of neurotransmitters on the ion channels in Deiters' cells. MATERIALS AND METHOD: Deiters' cells were isolated from guinea pig organs of Corti using collagenase and pipettes. Whole-cell patch clamps were performed under an inverted microscope and the current was measured with pClamp 8.0.2 software. RESULTS: The resting membrane potential was -21.1+/-3.5 mV. ATP (100 microM) treatment depolarized the potential to -3.1+/-1.1 mV, while the same concentration of Ach had no effect on the resting potential. In the voltage-clamping condition, the holding potential was 0 mV, and then a -80 mV pre-pulse was applied for 500 ms, followed by step pulses from -140 to +10 mV. Under these conditions, 10 microM ATP increased the inward current from -14.9+/-1.9 to -163.5+/-14.9 pA/pF at the maximal stimulus of -140 mV (n=4). In the current-voltage curve, the reversal potential was around -20 mV. Neither Ach nor carbachol induced current responses. The co-application of suramin (30 microM) and ATP (10 microM) suppressed the ATP-induced currents by 50%, and 30 microM of PPADS (pyridoxal-phosphate- 6-azophenyl-2, 4-disulphonic acid) inhibited the current almost to the level of the control. The purinoceptor-agonist, alpha, beta-meATP (alpha, beta-methylene adenosine triphosphate), 30 microM increased the inward current from -16.2+/-2.9 to -27.7+/-3.8 pA/pF, which was much smaller than the ATP-induced change. CONCLUSION: ATP-gated purinergic receptors may play an important role in regulating sound transduction by inducing an inward current and depolarizing the Deiters' cell membrane.

Changes in the Rate of Renin Secretion During Cell Cycle of As 4.1 Cells / 대한신장학회잡지

BACKGROUND: Renin is secreted from the juxtaglomerular (JG) cells in response to a wide variety of extracellular stimuli. To study the underlying mechanism of regulation of renin secretion at molecular level, pure JG cell lines (As 4.1) cloned from renal JG tumor was used. In this study, to explore the feasibility of As 4.1 cells as an in vitro model for renin secretion, the changes of renin secretion from As 4.1 in culture during cell cycle were characterized. METHODS: To address this issue, As 4.1s were synchronized in G0, G1, S, G2, early M and late M phase during experiment. RESULTS: The rate of renin secretion was above 1 ng AI/well/hr in G0, G2/M and early mitotic phase and 0.5 ng AI/well/hr in G1, G1/S, S and late mitotic phase. ML-7 (6x10(-5) M), an inhibitor of MLCK which is known to stimulate renin secretion, increased the rate of renin secretion much greater in G1, G1/S, S and late M phase than the other phases; in particular, in early mitotic phase it had no stimulation. On the other hand, the rate of renin secretion was not influenced through out cell cycles by calyculin A, an inhibitor of type 1 protein phosphatase. Forskolin, an activator of adenlyate cyclase resulting in an elevation of intracellular cyclic AMP, stimulated renin secretion only in S phase in a concentration dependent manner. CONCLUSION: The present study demonstrated that As 4.1 cells in culture secrete active renin in much the similar manner to JG cells in situ but its rate varies during each phase of the cell cycle. Thus As 4.1 cells can be utilized as an in vitro model for renin secretion. But, changes in the rate of renin secretion and the secretory responses to stimulators or inhibitors during cell cycle must be considered in conducting experiments to elucidate the cellular and molecular mechanism of the renin secretion.

Outward Rectifying Current in Isolated Deiters' Cells from Guinea Pig Cochlea / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The Deiters' cell is one of the supporting cells in the organ of Corti and is known to possibly regulate the signal transduction pathway in the organ of Corti. The signal transduction process can be modulated by ATP and acetylcholine, the so-called neurotransmitters, in Deiters' cells. Intracellular Ca2+ concentration can be also increased by these neurotransmitters and the control mechanism on the organ of Corti is highly suggested in Deiters' cells. Potassium ion (K+) is known to be important both in hair cells and supporting cells. Through K+ channel, the membrane potential may be controlled and the signal transduction pathway can be regulated. Furthermore, the motility of outer hair cell and the signal transduction from the apical stereocilia are considered to be regulated by this channel. The aim of this study is to record the K+ current in the isolated Deiters' cells from guinea pig cochlea. MATERIALS AND METHODS: Deiters' cells were isolated from the organ of Corti of guinea pig by using collagenase and a pipet. A whole cell patch clamp was performed under the inverted microscope and the current was measured with List-7 amplifier and pClamp 8.0.2 software. RESULTS: The resting membrane potential was -15.02+/-2.66 mV (n=6). When the cell membrane was hyperpolarized into -110 mV from the -40 mV holding potential, the peak current was -227+/-39.9 pA (n=15). After having depolarized to the maximum, (50 mV), the peak current was 7123+/-737 pA, and the reversal potentials of different external K+ concentration changed in the K+-dependent manner. About 80% of this current was inhibited by TEA. When K+ was substituted by Cs+, the peak current was 1788+/-231 pA at 50 mV step pulse. Activation curve of this outward current showed two different Vh (half activation voltage) and K (slope factor). CONCLUSION: Outward rectifying K+ channels exist in Deiters' cells and they can be inhibited by TEA and permeable to Cs+. More than two types of K+ current can exist and they may play a role in the recovery of membrane potential after depolarization,

The Characteristics of Spontaneous Action Potential of Cardiac Myocytes in Rabbit Pulmonary Veins

BACKGROUND AND OBJECTIVES: Atrial fibrillation is one of the most prevalent arrhythmia with clinical significance. Recently, some subset of paroxysmal atrial fibrillation was reported to be originated from a focal, rapidly firing source inside the large thoracic veins, such as pulmonary veins, superior vena cava and coronary sinus. The pulmonary veins are known to be the most frequent source of this type of atrial fibrillation. The proximal segment of pulmonary vein was reported to be made up with cardiac muscle cells. This study was performed 1)to define the characteristics of action potential of cardiac myocytes inside the rabbit pulmonary vein in single cell preparation, 2)to observe the changes in action potential and current activation to acetylcholine and isoproterenol, and 3)to compare these changes with those in atrial myocytes. METHOD AND RESULTS: In most of rabbit specimens, myocardial tissue extended over the pulmonary vein for a few millimeters(1-2.5mm). Single atrial myocyte and myocyte in pulmonary vein were successfully isolated. With using whole cell patch clamp technique, spontaneous activities of action potentials(APs) with diastolic depolarization were observed in 75% of pulmonary vein myocytes, in contrast to the absence of spontaneous activity in atrial myocytes. During spontaneous APs of pulmonary vein myocytes, the maximal diastolic potential was -50.5+/-6.5 mV and peak potential was 32.5+/-9.5 mV, and the frequency of APs was 1-2.5 Hz. During perfusion of isolated pulmonary vein myocytes with acetylcholine, resting membrane potential was hyperpolarized and spontaneous APs activity was markedly reduced or completely disappeared. These effects were observed in very low concentration of acetylcholine, even with 1-2 nM. The analysis of change of currents by applying step pulse revealed this response was mediated by activation of IK(ACh) and the current change was more prominent in pulmonary vein myocytes than atrial myocytes. The responses of these cells to isoproterenol were variable from increased spontaneous APs to inhibition of APs. CONCLUSION: This study revealed that pulmonary vein myocytes was another automatic pacemaking focus, same as sinoatrial nodal and Purkinje cells. These characteristics explain why focal atrial fibrillation was frequently initiated inside pulmonary veins.

Vesicular transport as a new paradigm in short-term regulation of transepithelial transport

The vectorial transepithelial transport of water and electrolytes in the renal epithelium is achieved by the polarized distribution of various transport proteins in the apical and basolateral membrane. The short-term regulation of transepithelial transport has been traditionally thought to be mediated by kinetic alterations of transporter without changing the number of transporters. However, a growing body of recent evidence supports the possibility that the stimulus-dependent recycling of transporter-carrying vesicles can alter the abundance of transporters in the plasma membrane in parallel changes in transepithelial transport functions. The abundance of transporters in the plasma membrane is determined by net balance between stimulus-dependent exocytic insertion of transporters into and endocytic retrieval of them from the plasma membrane. The vesicular recycling occurs along the tracts of the actin microfilaments and microtubules with associated motors. This review is to highlight the importance of vesicular transport in the short-term regulatory process of transepithelial transport in the renal epithelium. In the short-term regulation of many other renal transporters, vesicular transport is likely to be also involved. Thus, vesicular transport is now emerged as a wide-spread general regulatory mechanism involved in short-term regulation of renal functions.