Exploration of the Parameter Space of an Ion Channel Kinetic Model by a Markov-Chain-Based Methodology.

In this work, we propose a methodology based on Monte Carlo Markov chains to explore the parameter space of kinetic models for ion channels. The methodology allows the detection of potential parameter sets of a model that are compatible with experimentally obtained whole-cell currents, which could remain hidden when methods focus on obtaining the parameters that provide the best fit. To show its implementation and utility, we considered a four-state kinetic model proposed in the literature to describe the activation of the voltage-gated proton channel (Hv1), Biophysical Journal, 2014, 107, 1564. In that work, a set of values for the rate transitions that describe the channel kinetics at different intracellular H+ concentration (pHi) were obtained by the Simplex method. With our approach, we find that, in fact, there is more than one parameter set for each pHi, which renders the same open probability temporal course within the experimental error margin for all of the considered voltages. The large differences that we obtained for the values of some rate constants among the different solutions show that there is more than one possible interpretation of this channel behavior as a function of pHi. We also simulated a proposed new experimental condition where it is possible to observe that different sets of parameters yield different results. Our study highlights the importance of a comprehensive analysis of parameter space in kinetic models and the utility of the proposed methodology for finding potential solutions.

Novel Dimeric hHv1 Model and Structural Bioinformatic Analysis Reveal an ATP-Binding Site Resulting in a Channel Activating Effect.

The human voltage-gated proton channel (hHv1) is a highly selective ion channel codified by the HVCN1 gene. It plays a fundamental role in several physiological processes such as innate and adaptive immunity, insulin secretion, and sperm capacitation. Moreover, in humans, a higher hHv1 expression/function has been reported in several types of cancer cells. Here we report a multitemplate homology model of the hHv1 channel, built and refined as a dimer in Rosetta. The model was then subjected to extensive Gaussian accelerated molecular dynamics (GaMD) for enhanced conformational sampling, and representative snapshots were extracted by clustering analysis. Combining different structure- and sequence-based methodologies, we predicted a putative ATP-binding site located on the intracellular portion of the channel. Furthermore, GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal segments. According to the in silico predictions, we found that 3 mM intracellular ATP significantly increases the H+ current mediated by the hHv1 channel expressed in HEK293 cells and measured by the patch-clamp technique in an inside-out configuration (2.86 ± 0.63 fold over control at +40 mV). When ATP was added on the extracellular side, it was not able to activate the channel supporting the idea that the ATP-binding site resides in the intracellular face of the hHV1 channel. In a physiological and pathophysiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation, such as pancreatic ß-cells, immune cells, and cancer cells.

Determinants of Ca2+ release restitution: Insights from genetically altered animals and mathematical modeling.

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca2+ release restitution (CRR), and its alterations are potential triggers of Ca2+ arrhythmias. Although the control of CRR has been associated with SR Ca2+ load and RYR2 Ca2+ sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca2+ content versus the velocity of SR Ca2+ refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca2+ (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S1/S2) protocol. CRR and ICaL restitution increased as a function of the (S2-S1) coupling interval, following an exponential curve. When SR Ca2+ load was increased by increasing extracellular [Ca2+] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca2+]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca2+ load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca2+] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca2+ load.

Ablation of phospholamban rescues reperfusion arrhythmias but exacerbates myocardium infarction in hearts with Ca2+/calmodulin kinase II constitutive phosphorylation of ryanodine receptors.

AIMS: Abnormal Ca2+ release from the sarcoplasmic reticulum (SR), associated with Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of RyR2 at Ser2814, has consistently been linked to arrhythmogenesis and ischaemia/reperfusion (I/R)-induced cell death. In contrast, the role played by SR Ca2+ uptake under these stress conditions remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to attenuate reperfusion arrhythmias and cardiac injury elicited by increased RyR2-Ser2814 phosphorylation. METHODS AND RESULTS: We used WT mice, which have been previously shown to exhibit a transient increase in RyR2-Ser2814 phosphorylation at the onset of reperfusion; mice with constitutive pseudo-phosphorylation of RyR2 at Ser2814 (S2814D) to exacerbate CaMKII-dependent reperfusion arrhythmias and cardiac damage, and phospholamban (PLN)-deficient-S2814D knock-in (SDKO) mice resulting from crossbreeding S2814D with phospholamban knockout deficient (PLNKO) mice. At baseline, S2814D and SDKO mice had structurally normal hearts. Moreover none of the strains were arrhythmic before ischaemia. Upon cardiac I/R, WT, and S2814D hearts exhibited abundant arrhythmias that were prevented by PLN ablation. In contrast, PLN ablation increased infarct size compared with WT and S2814D hearts. Mechanistically, the enhanced SR Ca2+ sequestration evoked by PLN ablation in SDKO hearts prevented arrhythmogenic events upon reperfusion by fragmenting SR Ca2+ waves into non-propagated and non-arrhythmogenic events (mini-waves). Conversely, the increase in SR Ca2+ sequestration did not reduce but rather exacerbated I/R-induced SR Ca2+ leak, as well as mitochondrial alterations, which were greatly avoided by inhibition of RyR2. These results indicate that the increase in SR Ca2+ uptake is ineffective in preventing the enhanced SR Ca2+ leak of PLN ablated myocytes from either entering into nearby mitochondria and/or activating additional CaMKII pathways, contributing to cardiac damage. CONCLUSION: Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by CaMKII-dependent phosphorylation of RyR2-induced SR Ca2+ leak. These findings underscore the benefits of increasing SERCA2a activity in the face of SR Ca2+ triggered arrhythmias. However, enhanced SERCA2a cannot prevent but rather exacerbates I/R cardiac injury.

Experimental assessment of a myocyte-based multiscale model of cardiac contractile dysfunction.

Cardiac contractile dysfunction (CD) is a multifactorial syndrome caused by different acute or progressive diseases which hamper assessing the role of the underlying mechanisms characterizing a defined pathological condition. Mathematical modeling can help to understand the processes involved in CD and analyze their relative impact in the overall response. The aim of this study was thus to use a myocyte-based multiscale model of the circulatory system to simulate the effects of halothane, a volatile anesthetic which at high doses elicits significant acute CD both in isolated myocytes and intact animals. Ventricular chambers built using a human myocyte model were incorporated into a whole circulatory system represented by resistances and capacitances. Halothane-induced decreased sarco(endo)plasmic reticulum Ca2+ (SERCA2a) reuptake pump, transient outward K+ (Ito), Na+-Ca2+ exchanger (INCX) and L-type Ca2+ channel (ICaL) currents, together with ryanodine receptor (RyR2) increased open probability (Po) and reduced myofilament Ca2+ sensitivity, reproduced equivalent decreased action potential duration at 90% repolarization and intracellular Ca2+ concentration at the myocyte level reported in the literature. In the whole circulatory system, model reduction in mean arterial pressure, cardiac output and regional wall thickening fraction was similar to experimental results in open-chest sheep subjected to acute halothane overdose. Effective model performance indicates that the model structure could be used to study other changes in myocyte targets eliciting CD.

Nitration of nitrobenzene in Fenton's processes.

Previous studies of nitrobenzene (NB) degradation by Fenton and photo-Fenton technologies have demonstrated the formation and accumulation of 1,3-dinitrobenzene (1,3-DNB) as a highly toxic reaction intermediate. In the present study, we analyze the conditions that favor 1,3-DNB formation during NB degradation by Fe(2+)/H(2)O(2), Fe(3+)/H(2)O(2), UV/Fe(3+)/H(2)O(2) or UV/H(2)O(2) processes. Nitration yields in Fenton, Fenton-like and photo-Fenton techniques were much higher than those observed in UV/H(2)O(2) systems. Besides, several tests showed that 1,3-DNB formation increases with the initial iron concentration and decreases as the initial H(2)O(2) concentration increases. In order to asses the key species involved in NB nitration mechanism, additional experiments were performed in the presence of NO(2)(-)or NO(3)(-). In dark systems, 1,3-DNB yield significantly increased with increasing [NO(2)(-)]_(0), while it was not affected by the presence of NO(3)(-). In contrast, 1,3-DNB yields were higher and more strongly affected by the additive concentration in UV/NO(3)(-) systems than in UV/HNO(2)/NO(2)(-) systems. Dark experiments performed at pH 1.5 in excess of HNO(2) along with UV/NO(3)(-) tests conducted in the presence of 2-propanol show that hydroxyl radicals play an important role in NB nitration since NB molecule does not react with the nitrating agents ONOOH, .NO or .NO(2). The results indicate that, in the experimental domain tested, the prevailing NB nitration pathway involves the reaction between the .OH-NB adduct and .NO(2) radicals.

Kinetics of nitrobenzene and 4-nitrophenol degradation by UV irradiation in the presence of nitrate and nitrite ions.

In the present work we analyze the degradation rates of nitrobenzene (NBE) and 4-nitrophenol (PNP), by using UV irradiation in the presence of HNO(2)/NO(2)(-) or NO(3)(-) added as sources of hydroxyl radicals. With both nitroaromatic substrates pseudo-first-order kinetics were observed for conversion degrees of at least 45% within the analyzed experimental domain. The apparent rate constants (k(app)) were higher for UV/NO(3)(-) systems than for UV/HNO(2)/NO(2)(-) systems at high additive concentrations, whereas the opposite trend was observed for low additive concentrations. In addition, k(app) values were found to decrease with increasing substrate concentration. The analysis of the distribution of the most important products detected by HPLC suggests that reactions involving nitrogen species are likely to play a secondary role in the apparent rate constants measured. NBE and PNP degradation rates induced by HNO(2)/NO(2)(-) photolysis increase to a maximum value and then decrease with increasing additive concentration. On the other hand, substrate degradation rates increase with NO(3)(-) concentrations until a plateau is reached, but no decrease of k(app) is observed even at very high additive concentrations. The results may be quantitatively described by a simplified model that considers two opposite effects. On the one hand, the increase of additive concentrations increases the amount of photons absorbed by the photoactive species in both systems. On the other hand, high additive concentrations significantly decrease the degradation rates in UV/HNO(2)/NO(2)(-) systems where HNO(2) and NO(2)(-) are efficient hydroxyl radical scavengers, whereas this decrease is not observed in UV/NO(3)(-) systems since the scavenging ability of NO(3)(-) is much lower. Inner filter and scavenging effects are discussed and the results are compared with the ones previously reported for UV/H(2)O(2) systems.