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Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type Ca V 1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in Ca V 1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the Ca V 1.2 channel pore-forming subunit (α1 C ) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in Ca V 1.2 channel activity were associated with PKA activity, leading to α1 C phosphorylation at Ser 1928 Compared to arteries from low-fat diet (LFD)-fed mice and nondiabetic patients, arteries from high-fat diet (HFD)-fed mice and from diabetic patients had increased Ser 1928 phosphorylation and Ca V 1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser 1928 phosphorylation and Ca V 1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser 1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a Ca V 1.2 with Ser 1928 mutated to alanine (S1928A) lacked glucose-mediated increases in Ca V 1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in Ca V 1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1 C phosphorylation at Ser 1928 in stimulating Ca V 1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes., (Copyright © 2017, American Association for the Advancement of Science.)

Population-based computational approaches have been developed in recent years and helped to gain insight into arrhythmia mechanisms, and intra- and inter-patient variability (e.g., in drug responses). Here, we illustrate the use of multivariable logistic regression to analyze the factors that enhance or reduce the susceptibility to cellular arrhythmogenic events. As an example, we generate 1000 model variants by randomly modifying ionic conductances and maximal rates of ion transports in our atrial myocyte model and simulate an arrhythmia-provoking protocol that enhances early afterdepolarization (EAD) proclivity. We then treat EAD occurrence as a categorical, yes or no variable, and perform logistic regression to relate perturbations in model parameters to the presence/absence of EADs. We find that EAD formation is sensitive to the conductance of the voltage-gated Na + , the acetylcholine-sensitive and ultra-rapid K + channels, and the Na + /Ca 2+ exchange current, which matches our mechanistic understanding of the process and preliminary sensitivity analysis. The described technique: •allows investigating the factors underlying dichotomous outcomes, and is therefore a useful tool improve our understanding of arrhythmic risk;•is valid for analyzing both deterministic and stochastic models, and various phenomena (e.g., delayed afterdepolarizations and Ca 2+ sparks);•is computationally more efficient than one-at-a-time parameter sensitivity analysis.

Background: We have previously shown that non-equilibrium Na(+) current (INa) reactivation drives isoproterenol-induced phase-3 early afterdepolarizations (EADs) in mouse ventricular myocytes. In these cells, EAD initiation occurs secondary to potentiated sarcoplasmic reticulum Ca(2+) release and enhanced Na(+)/Ca(2+) exchange (NCX). This can be abolished by tetrodotoxin-blockade of INa, but not ranolazine, which selectively inhibits ventricular late INa., Aim: Since repolarization of human atrial myocytes is similar to mouse ventricular myocytes in that it is relatively rapid and potently modulated by Ca(2+), we investigated whether similar mechanisms can evoke EADs in human atrium. Indeed, phase-3 EADs have been shown to re-initiate atrial fibrillation (AF) during autonomic stimulation, which is a well-recognized initiator of AF., Methods: We integrated a Markov model of INa gating in our human atrial myocyte model. To simulate experimental results, we rapidly paced this cell model at 10Hz in the presence of 0.1μM acetylcholine and 1μM isoproterenol, and assessed EAD occurrence upon return to sinus rhythm (1Hz)., Results: Cellular Ca(2+) loading during fast pacing results in a transient period of hypercontractility after return to sinus rhythm. Here, fast repolarization and enhanced NCX facilitate INa reactivation via the canonical gating mode (i.e., not late INa burst mode), which drives EAD initiation. Simulating ranolazine administration reduces atrial peak INa and leads to faster repolarization, during which INa fails to reactivate and EADs are prevented., Conclusions: Non-equilibrium INa reactivation can critically contribute to arrhythmias, specifically in human atrial myocytes. Ranolazine might be beneficial in this context by blocking peak (not late) atrial INa., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca(2+) transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by β-adrenergic stimulation (β-AS). β-AS effect on sarcoplasmic reticulum Ca(2+) handling, Ca(2+), K(+) and Cl(-) currents, and Na(+)/K(+)-ATPase properties was included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca(2+) sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental β-AS responses on action potentials and Ca(2+) transient amplitude and kinetics. It also replicated the behavior of force-Ca(2+), release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The β-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing β-AS effects on L-type Ca(2+) channels or phospholamban limited Ca(2+) transients and contractile responses in parallel, while blocking phospholemman and K(+) channel (IKs) effects enhanced Ca(2+) and inotropy. Removal of β-AS effects from XBCa enhanced contractile force while decreasing peak Ca(2+) (due to greater Ca(2+) buffering), but had less effect on shortening. Conversely, preventing β-AS effects on XBcy preserved Ca(2+) transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of β-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. β-AS effects on XBCa and XBcy have greater impact on isometric and isotonic contraction, respectively., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The cardiac voltage gated Ca(2+) current (ICa) is critical to the electrophysiological properties, excitation-contraction coupling, mitochondrial energetics, and transcriptional regulation in heart. Thus, it is not surprising that cardiac ICa is regulated by numerous pathways. This review will focus on changes in ICa that occur during the cardiac action potential (AP), with particular attention to Ca(2+)-dependent inactivation (CDI), Ca(2+)-dependent facilitation (CDF) and how calmodulin (CaM) and Ca(2+)-CaM dependent protein kinase (CaMKII) participate in the regulation of Ca(2+) current during the cardiac AP. CDI depends on CaM pre-bound to the C-terminal of the L-type Ca(2+) channel, such that Ca(2+) influx and Ca(2+) released from the sarcoplasmic reticulum bind to that CaM and cause CDI. In cardiac myocytes CDI normally pre-dominates over voltage-dependent inactivation. The decrease in ICa via CDI provides direct negative feedback on the overall Ca(2+) influx during a single beat, when myocyte Ca(2+) loading is high. CDF builds up over several beats, depends on CaMKII-dependent Ca(2+) channel phosphorylation, and results in a staircase of increasing ICa peak, with progressively slower inactivation. CDF and CDI co-exist and in combination may fine-tune the ICa waveform during the cardiac AP. CDF may partially compensate for the tendency for Ca(2+) channel availability to decrease at higher heart rates because of accumulating inactivation. CDF may also allow some reactivation of ICa during long duration cardiac APs, and contribute to early afterdepolarizations, a form of triggered arrhythmias.

Sarcoplasmic reticulum (SR) Ca(2+) release mediates excitation–contraction coupling (ECC) in cardiac myocytes. It is triggered upon membrane depolarization by entry of Ca(2+) via L-type Ca(2+) channels (LTCCs), which undergo both voltage- and Ca(2+)-dependent inactivation (VDI and CDI, respectively). We developed improved models of L-type Ca(2+) current and SR Ca(2+) release within the framework of the Shannon-Bers rabbit ventricular action potential (AP) model. The formulation of SR Ca(2+) release was modified to reproduce high ECC gain at negative membrane voltages. An existing LTCC model was extended to reflect more faithfully contributions of CDI and VDI to total inactivation. Ba(2+) current inactivation included an ion-dependent component (albeit small compared with CDI), in addition to pure VDI. Under physiological conditions (during an AP) LTCC inactivates predominantly via CDI, which is controlled mostly by SR Ca(2+) release during the initial AP phase, but by Ca(2+) through LTCCs for the remaining part. Simulations of decreased CDI or K(+) channel block predicted the occurrence of early and delayed after depolarizations. Our model accurately describes ECC and allows dissection of the relative contributions of different Ca(2+) sources to total CDI, and the relative roles of CDI and VDI, during normal and abnormal repolarization.

Inactivation of L-type Ca channels (LTCC) is regulated by both Ca and voltage-dependent processes (CDI and VDI). To differentiate VDI and CDI, several experimental and theoretical studies have considered the inactivation of Ba current through LTCC (I(Ba)) as a measure of VDI. However, there is evidence that Ba can weakly mimic Ca, such that I(Ba) inactivation is still a mixture of CDI and VDI. To avoid this complication, some have used the monovalent cation current through LTCC (I(NS)), which can be measured when divalent cation concentrations are very low. Notably, I(NS) inactivation rate does not depend on current amplitude, and hence may reflect purely VDI. However, based on analysis of existent and new data, and modeling, we find that I(NS) can inactivate more rapidly and completely than I(Ba), especially at physiological temperature. Thus VDI that occurs during I(Ba) (or I(Ca)) must differ intrinsically from VDI during I(NS). To account for this, we have extended a previously published LTCC mathematical model of VDI and CDI into an excitation-contraction coupling model, and assessed whether and how experimental I(Ba) inactivation results (traditionally used in VDI experiments and models) could be recapitulated by modifying CDI to account for Ba-dependent inactivation. Thus, the view of a slow and incomplete I(NS) inactivation should be revised, and I(NS) inactivation is a poor measure of VDI during I(Ca) or I(Ba). This complicates VDI analysis experimentally, but raises intriguing new questions about how the molecular mechanisms of VDI differ for divalent and monovalent currents through LTCCs., (2010 Elsevier Ltd. All rights reserved.)

Rationale: Sodium channel blockers are used as gene-specific treatments in long-QT syndrome type 3, which is caused by mutations in the sodium channel gene (SCN5A). Response to treatment is influenced by biophysical properties of mutations., Objective: We sought to investigate the unexpected deleterious effect of mexiletine in a mutation combining gain-of- function and trafficking abnormalities., Methods and Results: A long-QT syndrome type 3 child experienced paradoxical QT prolongation and worsening of arrhythmias after mexiletine treatment. The SCN5A mutation F1473S expressed in HEK293 cells presented a right-ward shift of steady-state inactivation, enlarged window current, and huge sustained sodium current. Unexpectedly, it also reduced the peak sodium current by 80%. Immunostaining showed that mutant Nav1.5 is retained in the cytoplasm. Incubation with 10 micromol/L mexiletine rescued the trafficking defect of F1473S, causing a significant increase in peak current, whereas sustained current was unchanged. Using a Markovian model of the Na channel and a model of human ventricular action potential, we showed that simulated exposure of F1473S to mexiletine paradoxically increased action potential duration, mimicking QT prolongation seen in the index patient on mexiletine treatment., Conclusions: Sodium channel blockers are largely used to shorten QT intervals in carriers of SCN5A mutations. We provided evidence that these agents may facilitate trafficking of mutant proteins, thus exacerbating QT prolongation. These data suggest that caution should be used when recommending this class of drugs to carriers of mutations with undefined electrophysiological properties.