In silico Cell Therapy Model Restores Failing Human Myocyte Electrophysiology and Calcium Cycling in Fibrotic Myocardium

Myocardial delivery of human c-kit+ cardiac interstitial cells (hCICs) and human mesenchymal stem cells (hMSCs), an emerging approach for treating the failing heart, has been limited by an incomplete understanding of the effects on host myocardium. This computational study aims to model hCIC and hMSC effects on electrophysiology and calcium cycling of healthy and diseased human cardiomyocytes (hCM), and reveals a possible cardiotherapeutic benefit independent of putative regeneration processes. First, we developed an original hCIC mathematical model with an electrical profile comprised of distinct experimentally identified ion currents. Next, we verified the model by confirming it is representative of published experiments on hCIC whole-cell electrophysiology and on hCIC co-cultures with rodent cardiomyocytes. We then used our model to compare electrophysiological effects of hCICs to other non-excitable cells, as well as clinically relevant hCIC-hMSC combination therapies and fused hCIC-hMSC CardioChimeras. Simulation of direct coupling of hCICs to healthy or failing hCMs through gap junctions led to greater increases in calcium cycling with lesser reductions in action potential duration (APD) compared with hMSCs. Combined coupling of hCICs and hMSCs to healthy or diseased hCMs led to intermediate effects on electrophysiology and calcium cycling compared to individually coupled hCICs or hMSCs. Fused hCIC-hMSC CardioChimeras decreased healthy and diseased hCM APD and calcium transient amplitude compared to individual or combined cell treatments. Finally, to provide a theoretical basis for optimizing cell-based therapies, we randomized populations of 2,500 models incorporating variable hMSC and hCIC interventions and simulated their effects on restoring diseased cardiomyocyte electrophysiology and calcium handling. The permutation simulation predicted the ability to correct abnormal properties of heart failure hCMs in fibrotic, but not non-fibrotic, myocardium. This permutation experiment also predicted paracrine signaling to be a necessary and sufficient mechanism for this correction, counteracting the fibrotic effects while also restoring arrhythmia-related metrics such as upstroke velocity and resting membrane potential. Altogether, our in silico findings suggest anti-fibrotic effects of paracrine signaling are critical to abrogating pathological cardiomyocyte electrophysiology and calcium cycling in fibrotic heart failure, and support further investigation of delivering an optimized cellular secretome as a potential strategy for improving heart failure therapy.

Inhibition of Cdk5 in PV Neurons Reactivates Experience-Dependent Plasticity in Adult Visual Cortex

Cyclin-dependent kinase 5 (Cdk5) has been shown to play a critical role in brain development, learning, memory and neural processing in general. Cdk5 is widely distributed in many neuron types in the central nervous system, while its cell-specific role is largely unknown. Our previous study showed that Cdk5 inhibition restored ocular dominance (OD) plasticity in adulthood. In this study, we specifically knocked down Cdk5 in different types of neurons in the visual cortex and examined OD plasticity by optical imaging of intrinsic signals. Downregulation of Cdk5 in parvalbumin-expressing (PV) inhibitory neurons, but not other neurons, reactivated adult mouse visual cortical plasticity. Cdk5 knockdown in PV neurons reduced the evoked firing rate, which was accompanied by an increment in the threshold current for the generation of a single action potential (AP) and hyperpolarization of the resting membrane potential. Moreover, chemogenetic activation of PV neurons in the visual cortex can attenuate the restoration of OD plasticity by Cdk5 inhibition. Taken together, our results suggest that Cdk5 in PV interneurons may play a role in modulating the excitation and inhibition balance to control the plasticity of the visual cortex.

Hyperexcitability of Sensory Neurons in Fragile X Mouse Model

Sensory hypersensitivity and somatosensory deficits represent the core symptoms of Fragile X syndrome (FXS). These alterations are believed to arise from changes in cortical sensory processing, while potential deficits in the function of peripheral sensory neurons residing in dorsal root ganglia remain unexplored. We found that peripheral sensory neurons exhibit pronounced hyperexcitability in Fmr1 KO mice, manifested by markedly increased action potential (AP) firing rate and decreased threshold. Unlike excitability changes found in many central neurons, no significant changes were observed in AP rising and falling time, peak potential, amplitude, or duration. Sensory neuron hyperexcitability was caused primarily by increased input resistance, without changes in cell capacitance or resting membrane potential. Analyses of the underlying mechanisms revealed reduced activity of HCN channels and reduced expression of HCN1 and HCN4 in Fmr1 KO compared to WT. A selective HCN channel blocker abolished differences in all measures of sensory neuron excitability between WT and Fmr1 KO neurons. These results reveal a hyperexcitable state of peripheral sensory neurons in Fmr1 KO mice caused by dysfunction of HCN channels. In addition to the intrinsic neuronal dysfunction, the accompanying paper examines deficits in sensory neuron association/communication with their enveloping satellite glial cells, suggesting contributions from both neuronal intrinsic and extrinsic mechanisms to sensory dysfunction in the FXS mouse model.

Structure and mechanism of NALCN-FAM155A-UNC79-UNC80 channel complex

NALCN channel mediates sodium leak currents and is important for maintaining proper resting membrane potential. NALCN and FAM155A form the core complex of the channel, the activity of which essentially depends on the presence of both UNC79 and UNC80, two auxiliary proteins. NALCN, FAM155A, UNC79, and UNC80 co-assemble into a large hetero-tetrameric channel complex. Genetic mutations of NALCN channel components lead to neurodevelopmental diseases. However, the structure and mechanism of the intact channel complex remain elusive. Here, we present the cryo-EM structure of the mammalian NALCN-FAM155A-UNC79-UNC80 quaternary complex. The structure showed that UNC79-UNC80 form a large piler-shaped heterodimer which was tethered to the intracellular side of the NALCN channel through tripartite interactions with the cytoplasmic loops of NALCN. Two interactions are essential for proper cell surface localization of NALCN. The other interaction relieves the self-inhibition of NALCN by pulling the auto-inhibitory CTD Interacting Helix (CIH) out of its binding site.

Membrane voltage fluctuations in human breast cancer cells

Cancer cells feature a resting membrane potential (Vm) that is depolarized compared to normal cells, and express active ionic conductances, which factor directly in their pathophysiological behavior. Despite similarities to 'excitable' tissues, relatively little is known about cancer cell Vm dynamics. With high-throughput, cellular-resolution Vm imaging, we characterized Vm fluctuations of hundreds of human triple-negative breast cancer MDA-MB-231 cells and compared to non-cancerous breast epithelial MCF-10A cells. By quantifying their Dynamic Electrical Signatures (DESs) through an unsupervised machine-learning protocol, we identified four classes ranging from "noisy" to "blinking/waving". The Vm of MDA-MB-231 cells exhibited spontaneous, transient hyperpolarizations that were inhibited by the voltage-gated sodium channel blocker tetrodotoxin. The Vm of MCF-10A cells was comparatively static, but fluctuations increased following treatment with transforming growth factor-β1, a canonical inducer of the epithelial-to-mesenchymal transition. These data suggest that the ability to generate Vm fluctuations is acquired during transformation and may participate in oncogenesis.

Ion Channels and Transporters in Muscle Cell Differentiation

Investigations on ion channels in muscle tissues have mainly focused on physiological muscle function and related disorders, but emerging evidence supports a critical role of ion channels and transporters in developmental processes, such as controlling the myogenic commitment of stem cells. In this review, we provide an overview of ion channels and transporters that influence skeletal muscle myoblast differentiation, cardiac differentiation from pluripotent stem cells, as well as vascular smooth muscle cell differentiation. We highlight examples of model organisms or patients with mutations in ion channels. Furthermore, a potential underlying molecular mechanism involving hyperpolarization of the resting membrane potential and a series of calcium signaling is discussed.

The hyperpolarization-activated current shifts the dynamic range of a voltage-dependent electrical synapse

Like their chemical counterparts, electrical synapses show complex dynamics such as rectification and voltage dependence that interact with other electrical processes in neurons. The consequences arising from these interactions for the electrical behavior of the synapse, and the dynamics they create, remain largely unexplored. Using a voltage-dependent electrical synapse between a descending modulatory projection neuron (MCN1) and a motor neuron (LG) in the crustacean stomatogastric ganglion, we find that the influence of the hyperpolarization-activated inward current (Ih) is critical to the function of the electrical synapse. When we blocked Ih with CsCl, the voltage dependence of the electrical synapse shifted by 18.7 mV to more hyperpolarized voltages, placing the dynamic range of the electrical synapse outside of the range of voltages used by the LG motor neuron (-60.2 mV to -44.9 mV). With dual electrode current- and voltage-clamp recordings, we demonstrate that this voltage shift is due to a sustained effect of Ih on the presynaptic MCN1 axon terminal membrane potential. Ih-induced depolarization of the axon terminal membrane potential increased the electrical postsynaptic potentials and currents. With Ih present, the axon terminal resting membrane potential depolarized, shifting the dynamic range of the electrical synapse towards the functional range of the motor neuron. We thus demonstrate that the function of an electrical synapse is critically influenced by a voltage-dependent ionic current (Ih).

Galvanotaxis of ciliates: Spatiotemporal dynamics of Coleps hirtus under electric fields

Galvanotaxis describes the functional response of organisms to electric fields. In ciliates, the electric field influences the electrophysiology and thus the cilia beat dynamics. This leads to a change of the swimming direction towards the cathode. The dynamical response to electric fields of Coleps hirtus has not been studied since the observations of Verworn in 1890 (1). While galvanotaxis has been studied in other cilitates, C. hirtus exhibit properties not found elsewhere, such as biomineralization-processes of alveolar plates with impact on the intracellular calcium regulation and a bimodal resting membrane potential, which leads unique electrophysiological driven bimodal swimming dynamics. Here, we statistically analyze the galvanotactic dynamics of C. hirtus by automated cell tracking routines. We found that the number of cells that show a galvanotactic response, increases with the increase of the applied electric field strength with a mean at about 2.1 V/cm. The spatiotemporal swimming dynamics change and lead to a statistical increase of linear elongated cell trajectories that point toward the cathode. Further, the increase of the electric fields decreases the mean velocity variance for electric fields larger than about 1.3 V/cm, while showing no significant change in the absolute velocity for any applied electric field. Fully functional galvanotactic responses were observed at a minimum extracellular calcium concentration of 20 μM. The results add important insights to the current understanding of cellular dynamics of ciliates and suggest that the currently accepted model lags the inclusion of the swimming dynamics and the complex calcium regulatory system of the cell. The results of this study do not only extend the fundamental understanding of C. hirtus dynamics, but also open possibilities for technical applications, such as biosensors or microrobots in the future.

Ia EPSPs in rat spinal motoneurons are potentiated after a 5-week whole-body vibration

Whole-body vibration (WBV) is often applied as an alternative method for strength training or to prevent muscle force decrease. Previous studies indicated that WBV induced: 1) changes in the contractile parameters predominantly of fast motor units, 2) higher motoneuron excitability, and 3) higher motoneuron firing rates at lower stimulus intensities compared with the control. In this study, we evaluated the influence of WBV on Ia monosynaptic input from muscle spindles because the tonic vibration reflex is responsible for the enhancement of muscle activity observed after WBV. The aim was to answer the question of whether repeated activation of muscle spindles during WBV may result in altered synaptic excitation of motoneurons. WBV was performed on adult male Wistar rats, 5 days per week, for 5 weeks, and each daily session consisted of four 30-s runs of vibration at 50 Hz. Fast-type medial gastrocnemius motoneurons were investigated intracellularly in deeply anesthetized animals in the experimental (n=7, 34 motoneurons) and control (n=7, 32 motoneurons) groups. Monosynaptic Ia EPSPs were evoked by electrical stimulation of afferent fibers from the synergistic lateral gastrocnemius and soleus muscles. Data were analyzed using a mixed linear model. WBV induced an increase of the mean EPSP amplitude by 28% (P=0.025), correlated with the resting membrane potential and input resistance, and a shortening of the mean EPSP rise time by 11% (P=0.012). The potentiation of synaptic excitation of motoneurons indicates that WBV may support rehabilitation or training processes aimed at increasing muscle strength on the basis of increased motoneuronal drive.