Shedding Light on Cardiac Excitation: In Vitro and In Silico Analysis of Native Ca2+ Channel Activation in Guinea Pig Cardiomyocytes using Organic Photovoltaic Devices.

OBJECTIVE: This study aims to explore the potential of organic electrolytic photocapacitors (OEPCs), an innovative photovoltaic device, in mediating the activation of native voltage-gated Cav1.2 channels (ICa,L) in Guinea pig ventricular cardiomyocytes. METHODS: Whole-cell patch-clamp recordings were employed to examine light-triggered OEPC mediated ICa,L activation, integrating the channel's kinetic properties into a multicompartment cell model to take intracellular ion concentrations into account. A multidomain model was additionally incorporated to evaluate effects of OEPC-mediated stimulation. The final model combines external stimulation, multicompartmental cell simulation, and a patch-clamp amplifier equivalent circuit to assess the impact on achievable intracellular voltage changes. RESULTS: Light pulses activated ICa,L, with amplitudes similar to voltage-clamp activation and high sensitivity to the L-type Ca2+ channel blocker, nifedipine. Light-triggered ICa,L inactivation exhibited kinetic parameters comparable to voltage-induced inactivation. CONCLUSION: OEPC-mediated activation of ICa,L demonstrates their potential for nongenetic optical modulation of cellular physiology potentially paving the way for the development of innovative therapies in cardiovascular health. The integrated model proves the light-mediated activation of ICa,L and advances the understanding of the interplay between the patch-clamp amplifier and external stimulation devices. SIGNIFICANCE: Treating cardiac conduction disorders by minimal-invasive means without genetic modifications could advance therapeutic approaches increasing patients' quality of life compared with conventional methods employing electronic devices.

Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node.

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Consequences of somatic mutations of GIRK1 detected in primary malign tumors on expression and function of G-protein activated, inwardly rectifying, K+ channels.

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.

Light Stimulation of Neurons on Organic Photocapacitors Induces Action Potentials with Millisecond Precision.

Nongenetic optical control of neurons is a powerful technique to study and manipulate the function of the nervous system. This research has benchmarked the performance of organic electrolytic photocapacitor (OEPC) optoelectronic stimulators at the level of single mammalian cells: human embryonic kidney (HEK) cells with heterologously expressed voltage-gated K+ channels and hippocampal primary neurons. OEPCs act as extracellular stimulation electrodes driven by deep red light. The electrophysiological recordings show that millisecond light stimulation of OEPC shifts conductance-voltage plots of voltage-gated K+ channels by ≈30 mV. Models are described both for understanding the experimental findings at the level of K+ channel kinetics in HEK cells, as well as elucidating interpretation of membrane electrophysiology obtained during stimulation with an electrically floating extracellular photoelectrode. A time-dependent increase in voltage-gated channel conductivity in response to OEPC stimulation is demonstrated. These findings are then carried on to cultured primary hippocampal neurons. It is found that millisecond time-scale optical stimuli trigger repetitive action potentials in these neurons. The findings demonstrate that OEPC devices enable the manipulation of neuronal signaling activities with millisecond precision. OEPCs can therefore be integrated into novel in vitro electrophysiology protocols, and the findings can inspire in vivo applications.

A549 in-silico 1.0: A first computational model to simulate cell cycle dependent ion current modulation in the human lung adenocarcinoma.

Lung cancer is still a leading cause of death worldwide. In recent years, knowledge has been obtained of the mechanisms modulating ion channel kinetics and thus of cell bioelectric properties, which is promising for oncological biomarkers and targets. The complex interplay of channel expression and its consequences on malignant processes, however, is still insufficiently understood. We here introduce the first approach of an in-silico whole-cell ion current model of a cancer cell, in particular of the A549 human lung adenocarcinoma, including the main functionally expressed ion channels in the plasma membrane as so far known. This hidden Markov-based model represents the electrophysiology behind proliferation of the A549 cell, describing its rhythmic oscillation of the membrane potential able to trigger the transition between cell cycle phases, and it predicts membrane potential changes over the cell cycle provoked by targeted ion channel modulation. This first A549 in-silico cell model opens up a deeper insight and understanding of possible ion channel interactions in tumor development and progression, and is a valuable tool for simulating altered ion channel function in lung cancer electrophysiology.

Hypochlorite-Modified LDL Induces Arrhythmia and Contractile Dysfunction in Cardiomyocytes.

Neutrophil-derived myeloperoxidase (MPO) and its potent oxidant, hypochlorous acid (HOCl), gained attention as important oxidative mediators in cardiac damage and dysfunction. As cardiomyocytes generate low-density lipoprotein (LDL)-like particles, we aimed to identify the footprints of proatherogenic HOCl-LDL, which adversely affects cellular signalling cascades in various cell types, in the human infarcted myocardium. We performed immunohistochemistry for MPO and HOCl-LDL in human myocardial tissue, investigated the impact of HOCl-LDL on electrophysiology and contractility in primary cardiomyocytes, and explored underlying mechanisms in HL-1 cardiomyocytes and human atrial appendages using immunoblot analysis, qPCR, and silencing experiments. HOCl-LDL reduced ICa,L and IK1, and increased INaL, leading to altered action potential characteristics and arrhythmic events including early- and delayed-afterdepolarizations. HOCl-LDL altered the expression and function of CaV1.2, RyR2, NCX1, and SERCA2a, resulting in impaired contractility and Ca2+ homeostasis. Elevated superoxide anion levels and oxidation of CaMKII were mediated via LOX-1 signaling in HL-1 cardiomyocytes. Furthermore, HOCl-LDL-mediated alterations of cardiac contractility and electrophysiology, including arrhythmic events, were ameliorated by the CaMKII inhibitor KN93 and the INaL blocker, ranolazine. This study provides an explanatory framework for the detrimental effects of HOCl-LDL compared to native LDL and cardiac remodeling in patients with high MPO levels during the progression of cardiovascular disease.

Modeling External Stimulation of Excitable Cells Using a Novel Light-Activated Organic Semiconductor Technology.

Optoelectronic neurostimulation is a promising, minimally invasive treatment modality for neuronal damage, in particular for patients with traumatic brain injury. In this work, a newly developed optoelectronic device, a so-called photocap, based on light-activated organic semiconductor structures with high spatial and temporal resolution is investigated. To prove and verify the feasibility of this new technology, a mathematical model was developed, simulating the electrical response of excitable cells to photocap stimulation. In the first step, a comprehensive technical review of the device concept was performed, building the basis for setting up the simulation model. The simulations demonstrate that photocaps may serve as a stimulation device, triggering action potentials in neural or cardiac cells. Our first results show that the model serves as a perfect tool for evaluating and further developing this new technology, showing high potential for introducing new and innovative therapy methods in the field of optoelectronic cell stimulation.

TRIC-A shapes oscillatory Ca2+ signals by interaction with STIM1/Orai1 complexes.

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A-mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A-dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release-activated Ca2+ channel 1 (Orai1) within ER-plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.

GIRK1 triggers multiple cancer-related pathways in the benign mammary epithelial cell line MCF10A.

Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Saxagliptin but Not Sitagliptin Inhibits CaMKII and PKC via DPP9 Inhibition in Cardiomyocytes.

Some oral anti-hyperglycemic drugs, including gliptins that inhibit dipeptidyl peptidase 4 (DPP4), have been linked to the increased risk of heart failure (HF) in type-2 diabetic patients. While the cardiovascular safety trial, TECOS, revealed no link between sitagliptin and the risk of HF, a substantial 27% increase in the hospitalization for HF was observed in type-2 diabetic patients treated with saxagliptin within the SAVOR-TIMI 53 trial. A previous in vitro study revealed that saxagliptin impairs the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-phospholamban (PLB)-sarcoplasmic reticulum Ca2+-ATPase 2a axis and protein kinase C (PKC) activity in cardiomyocytes leading to impaired cardiac contractility and electrophysiological function. However, the link between saxagliptin and its target proteins (CaMKII and PKC) remains to be explored. Since DPP8 and DPP9 (but not DPP4) are expressed by cardiomyocytes and saxagliptin is internalized by cardiomyocytes, we investigated whether DPP8/9 contribute to saxagliptin-mediated inhibition of CaMKII and PKC activity. Structural analysis revealed that the DPP4-saxagliptin interaction motif (S630, Y547) for the cyanopyrrolidine group is conserved in DPP8 (S755, Y669) and DPP9 (S730, Y644). Conversely, F357 that facilitates binding of the anchor lock domain of sitagliptin in the S2 extensive subsite of DPP4 is not conserved in DPP8/9. In parallel, unlike saxagliptin, sitagliptin did not affect phosphorylation of CaMKII/PLB or activity of PKC in HL-1 cardiomyocytes. These findings were recapitulated by pharmacological inhibition (TC-E-5007, a DPP8/9 antagonist) and knock-down of DPP9 (but not DPP8). In primary mouse ventricular cardiomyocytes, saxagliptin (but not sitagliptin) impaired Ca2+ transient relaxation and prolonged action potential duration (APD). These results suggest that saxagliptin-DPP9 interaction impairs the CaMKII-PLB and PKC signaling in cardiomyocytes. We reveal a novel and potential role of DPP9 in cardiac signaling. The interaction of saxagliptin with DPP9 may represent an underlying mechanism for the link between saxagliptin and HF. Elucidation of saxagliptin-DPP9 interaction and downstream events may foster a better understanding of the role of gliptins as modulators of cardiac signaling.

Sinoatrial Beat to Beat Variability Assessed by Contraction Strength in Addition to the Interbeat Interval.

Beat to beat variability of cardiac tissue or isolated cells is frequently investigated by determining time intervals from electrode measurements in order to compute scale dependent or scale independent parameters. In this study, we utilize high-speed video camera recordings to investigate the variability of intervals as well as mechanical contraction strengths and relative contraction strengths with nonlinear analyses. Additionally, the video setup allowed us simultaneous electrode registrations of extracellular potentials. Sinoatrial node tissue under control and acetylcholine treated conditions was used to perform variability analyses by computing sample entropies and Higuchi dimensions. Beat to beat interval variabilities measured by the two recording techniques correlated very well, and therefore, validated the video analyses for this purpose. Acetylcholine treatment induced a reduction of beating rate and contraction strength, but the impact on interval variability was negligible. Nevertheless, the variability analyses of contraction strengths revealed significant differences in sample entropies and Higuchi dimensions between control and acetylcholine treated tissue. Therefore, the proposed high-speed video camera technique might represent a non-invasive tool that allows long-lasting recordings for detecting variations in beating behavior over a large range of scales.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons.

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Dipeptidyl peptidase-4 independent cardiac dysfunction links saxagliptin to heart failure.

Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase-4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca2+/calmodulin-dependent protein kinase II-phospholamban-sarcoplasmic reticulum Ca2+-ATPase 2a axis and Na+-Ca2+ exchanger function in Ca2+ extrusion. This resulted in reduced sarcoplasmic reticulum Ca2+ content, diastolic Ca2+ overload, systolic dysfunction and impaired contractile force. Furthermore, saxagliptin reduced protein kinase C-mediated delayed rectifier K+ current that prolonged action potential duration and consequently QTc interval. Importantly, saxagliptin aggravated pre-existing cardiac dysfunction induced by ischemia/reperfusion injury. In conclusion, our novel results provide mechanisms for the off-target deleterious effects of saxagliptin on cardiac function and support the outcome of SAVOR-TIMI 53 trial that linked saxagliptin with the risk of heart failure.

I(f) blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes.

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.