Dengue virus infection is a global health problem for which there have been challenges to obtaining a cure. Current vaccines and anti-viral drugs can only be narrowly applied in ongoing clinical trials. We employed computational methods based on structure-function relationships between human host kinases and viral nonstructural protein 3 (NS3) to understand viral replication inhibitors’ therapeutic effect. Phosphorylation at each of the two most evolutionarily conserved sites of NS3, serine 137 and threonine 189, compared to the unphosphorylated state were studied with molecular dynamics and docking simulations. The simulations suggested that phosphorylation at serine 137 caused a more remarkable structural change than phosphorylation at threonine 189, specifically located at amino acid residues 49–95. Docking studies supported the idea that phosphorylation at serine 137 increased the binding affinity between NS3 and nonstructural Protein 5 (NS5), whereas phosphorylation at threonine 189 decreased it. The interaction between NS3 and NS5 is essential for viral replication. Docking studies with the antiviral plant flavonoid Quercetin with NS3 indicated that Quercetin physically occluded the serine 137 phosphorylation site. Taken together, these findings suggested a specific site and mechanism by which Quercetin inhibits dengue and possible other flaviviruses.

Calcium (Ca²⁺) plays a central role in the excitation and contraction of cardiac myocytes. Experiments have indicated that calcium release is stochastic and regulated locally suggesting the possibility of spatially heterogeneous calcium levels in the cells. This spatial heterogeneity might be important in mediating different signaling pathways. During more than 50 years of computational cell biology, the computational models have been advanced to incorporate more ionic currents, going from deterministic models to stochastic models. While periodic increases in cytoplasmic Ca²⁺ concentration drive cardiac contraction, aberrant Ca²⁺ release can underly cardiac arrhythmia. However, the study of the spatial role of calcium ions has been limited due to the computational expense of using a three-dimensional stochastic computational model. In this paper, we introduce a three-dimensional stochastic computational model for rat ventricular myocytes at the whole-cell level that incorporate detailed calcium dynamics, with (1) non-uniform release site placement, (2) non-uniform membrane ionic currents and membrane buffers, (3) stochastic calcium-leak dynamics and (4) non-junctional or rogue ryanodine receptors. The model simulates spark-induced spark activation and spark-induced Ca²⁺ wave initiation and propagation that occur under conditions of calcium overload at the closed-cell condition, but not when Ca²⁺ levels are normal. This is considered important since the presence of Ca²⁺ waves contribute to the activation of arrhythmogenic currents.

Cardiac alternans is characterized by alternating weak and strong beats of the heart. This signaling at the cellular level may appear as alternating long and short action potentials (APs) that occur in synchrony with alternating large and small calcium transients, respectively. Previous studies have suggested that alternans manifests itself through either a voltage dependent mechanism based upon action potential restitution or as a calcium dependent mechanism based on refractoriness of calcium release. We use a novel model of cardiac excitation-contraction (EC) coupling in the rat ventricular myocyte that includes 20,000 calcium release units (CRU) each with 49 ryanodine receptors (RyR2s) and 7 L-type calcium channels that are all stochastically gated. The model suggests that at the cellular level in the case of alternans produced by rapid pacing, the mechanism requires a synergy of voltage- and calcium-dependent mechanisms. The rapid pacing reduces AP duration and magnitude reducing the number of L-type calcium channels activating individual CRUs during each AP and thus increases the population of CRUs that can be recruited stochastically. Elevated myoplasmic and sarcoplasmic reticulum (SR) calcium, [Ca²⁺]_myo and [Ca²⁺]_SR respectively, increases ryanodine receptor open probability (P_o) according to our model used in this simulation and this increased the probability of activating additional CRUs. A CRU that opens in one beat is less likely to open the subsequent beat due to refractoriness caused by incomplete refilling of the junctional sarcoplasmic reticulum (jSR). Furthermore, the model includes estimates of changes in Na⁺ fluxes and [Na⁺]_i and thus provides insight into how changes in electrical activity, [Na⁺]_i and sodium-calcium exchanger activity can modulate alternans. The model thus tracks critical elements that can account for rate-dependent changes in [Na⁺]_i and [Ca²⁺]_myo and how they contribute to the generation of Ca²⁺ signaling alternans in the heart.

Calcium (Ca²⁺) sparks are the elementary events of excitation–contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca²⁺ ([Ca²⁺]_i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca²⁺ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca²⁺]_myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted “nonconducting” form of Ca²⁺-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca²⁺ fractional release was lower due to the smaller difference between diastolic and systolic [Ca²⁺]_SR. Restitution analysis through the S1S2 protocol and increased LCC Ca²⁺-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca²⁺ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca²⁺ release events and ion channel responses throughout various stimuli.

Cardiovascular disease is the leading cause of death worldwide due in a large part to arrhythmia. In order to understand how calcium dynamics play a role in arrhythmogenesis, normal and dysfunctional Ca²⁺ signaling in a subcellular, cellular, and tissued level is examined using cardiac ventricular myocytes at a high temporal and spatial resolution using multiscale computational modeling. Ca²⁺ sparks underlie normal excitation–contraction coupling. However, under pathological conditions, Ca²⁺ sparks can combine to form Ca²⁺ waves. These propagating elevations of (Ca²⁺)_i can activate an inward Na⁺–Ca²⁺ exchanger current (I_NCX) that contributes to early after-depolarization (EADs) and delayed after-depolarizations (DADs). However, how cellular currents lead to full depolarization of the myocardium and how they initiate extra systoles is still not fully understood. This study explores how many myocytes must be entrained to initiate arrhythmogenic depolarizations in biophysically detailed computational models. The model presented here suggests that only a small number of myocytes must activate in order to trigger an arrhythmogenic propagating action potential. These conditions were examined in 1-D, 2-D, and 3-D considering heart geometry. The depolarization of only a few hundred ventricular myocytes is required to trigger an ectopic depolarization. The number decreases under disease conditions such as heart failure. Furthermore, in geometrically restricted parts of the heart such as the thin muscle strands found in the trabeculae and papillary muscle, the number of cells needed to trigger a propagating depolarization falls even further to less than ten myocytes.

Calsequestrin Type 2 (CASQ2) is a high-capacity, low-affinity, Ca²⁺-binding protein expressed in the sarcoplasmic reticulum (SR) of the cardiac myocyte. Mutations in CASQ2 have been linked to the arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT2) that occurs with acute emotional stress or exercise can result in sudden cardiac death (SCD). CASQ2^G112+5X is a 16 bp (339–354) deletion CASQ2 mutation that prevents the protein expression due to premature stop codon. Understanding the subcellular mechanisms of CPVT2 is experimentally challenging because the occurrence of arrhythmia is rare. To obtain an insight into the characteristics of this rare disease, simulation studies using a local control stochastic computational model of the Guinea pig ventricular myocyte investigated how the mutant CASQ2s may be responsible for the development of an arrhythmogenic episode under the condition of β-adrenergic stimulation or in the slowing of heart rate afterward once β-adrenergic stimulation ceases. Adjustment of the computational model parameters based upon recent experiments explore the functional changes caused by the CASQ2 mutation. In the simulation studies under rapid pacing (6 Hz), electromechanically concordant cellular alternans appeared under β-adrenergic stimulation in the CPVT mutant but not in the wild-type nor in the non-β-stimulated mutant. Similarly, the simulations of accelerating pacing from slow to rapid and back to the slow pacing did not display alternans but did generate early afterdepolarizations (EADs) during the period of second slow pacing subsequent acceleration of rapid pacing.