KI Tarpląstelinės sąveikos tyrimų laboratorija (10.08.02)

Propofol is a widely used intravenous general anesthetic characterized by rapid induction and recovery. One of its major side effects is a reduction in systemic vascular resistance, leading to hypotension, and in some cases inhibition of cardiac conduction, resulting in bradycardia. Although these effects have been recognized for decades, the underlying molecular mechanisms remain incompletely understood. Propofol has been shown to modulate muscarinic receptors, sinoatrial node activity, and cardiac conduction in a concentration-dependent manner, while also exerting protective effects against ventricular arrhythmias during myocardial ischemia. Several studies suggest that propofol may modulate intercellular coupling via gap junction channels composed of connexin - 43 (Cx43), which play a critical role in vascular coordination and cardiac impulse propagation. The aim of this study was to investigate the effects of propofol on Cx43 gap junction channels. Gap junctional conductance was measured in HeLa cells expressing Cx43 using the dual whole-cell patch-clamp technique. Our results demonstrate that propofol significantly inhibits Cx43 gap junction channels, with complete inhibition observed at 45 µM. Previous studies have suggested that propofol modulates Cx43 gap junction channels via protein kinase C (PKC) activation, which phosphorylates Cx43 and reduces intercellular coupling. Consistent with these findings, we observed that a PKC-specific inhibitor significantly attenuated the inhibitory effect of propofol, confirming a PKC-dependent regulatory mechanism. To further investigate propofol’s mechanism of action, we performed experiments using a truncated Cx43 construct lacking the C-terminal domain, which is a primary site of phosphorylation involved in the regulation of channel opening and closing. Interestingly, our initial data show that 45 µM propofol still induced a complete blockade of these truncated channels. These findings suggest that propofol inhibits Cx43 gap junction channels through both indirect and direct mechanisms.

Įvadas. Širdies ir kraujagyslių ligos vis dar išlieka viena iš dažniausių mirties priežasčių pasaulyje. Šiuolaikinė medicina siekia mažinti širdies ir kraujagyslių sistemos sukeltų ligų skaičių, ieško efektyvesnių gydymo strategijų. Viena jų – ginkmedžio (lot. Ginkgo biloba) ekstrakto panaudojimas. Šio tyrimo tikslas buvo apžvelgti naujausią esamą mokslinę literatūrą, kurioje aprašoma ginkmedžio ekstrakto sudedamųjų dalių nauda širdies ir kraujagyslių sistemai bei išsiaiškinti, kokį toksinį poveikį gali turėti nesaugus ginkmedžio ekstrakto vartojimas. Tyrimo rezultatai atskleidė, kad tokios flavanoidų ir terpenoidų savybės, kaip oksidacinio streso mažinimas, uždegimo slopinimas, antimikrobinis poveikis ir kt. yra reikšmingos siekiant geresnio širdies ir kraujagyslių ligų gydymo rezultato. Neviršijant maksimalios rekomenduojamos ginkmedžio lapų ekstrakto paros dozės, šis papildas - gerai toleruojamas, turi mažai šalutinių reiškinių. Tikslas. Išnagrinėti ginkmedžio ekstrakto sudedamųjų dalių poveikį širdies ir kraujagyslių sistemai, bei išsiaiškinti toksinio poveikio mechanizmus. Metodai. PubMed duomenų bazėje rastos ir išnagrinėtos 29 publikacijos ir 1 internetinės prieigos šaltinis, atitinkantis temą. Rezultatai. Ginkgo biloba ekstraktas pasižymi reikšmingu antioksidaciniu, priešuždegiminiu, neuroprotekciniu ir antitromboziniu poveikiu, galinčiu padėti širdies ir kraujagyslių ligų profilaktikai bei gydymui. Pagrindinės ekstrakto veikliosios medžiagos – flavonoidai, terpenoidai ir ginkgolidai – gali gerinti kraujo tėkmę, apsaugoti nuo oksidacinio streso ir uždegimo bei koreguoti dislipidemiją. Toksinio poveikio rizika daugiausia siejama su ginkgolio rūgštimis bei sąveika su kitais vaistais, ypač antikoaguliantais ir antitrombocitiniais preparatais. Standartizuotas ekstraktas EGb 761 laikomas saugiu vartoti rekomenduojamomis dozėmis (iki 240 mg/d.), tačiau didelės rizikos grupės pacientams reikalingas papildomas krešėjimo rodiklių stebėjimas.

Connexin (Cx) hemichannels form intercellular gap junction channels but can also function independently. These large-pore channels are permeable not only to ions but also to small signalling molecules and metabolites. This functional property is relevant to many physiological processes and can be altered by various biochemical factors or disease-causing mutations. In this study, we present a methodology for quantifying and comparing the permeabilities of hemichannels formed by different Cx isoforms using a combination of fluorescence imaging, electrophysiological recordings and mathematical modelling. Fluorescence imaging, coupled with mathematical modelling based on Fick’s law and/or the Goldman-Hodgkin-Katz current equation, enables assessment of tracer diffusion rates. These data are integrated with independently obtained electrophysiological measurements of hemichannel activity into a unified statistical model based on the likelihood ratio test. Simulation-based analyses demonstrate that this approach can reliably detect differences as low as two-fold in hemichannel permeability using datasets of moderate size (n < 100). Crucially, this approach requires only a minimal amount of time-intensive electrophysiological recording and leverages higher-throughput fluorescence measurements, which can be further streamlined using computational tools for automated cell detection and data extraction. We apply this methodology to compare the permeability of hemichannels formed by wild-type Cx26 and a pore-lining variant, Cx26⁎A49E. Our results show a significant increase in DAPI permeability in Cx26⁎A49E hemichannels, consistent with previous findings. This methodology can be extended to assess the permeabilities of other large-pore channels.

Galvos smegenų insultas (GSI) išlieka viena iš dažniausių mirties ir negalios priežasčių pasaulyje [1]. Patikimi duomenys apie insulto sergamumo ir mirtingumo tendencijas yra svarbūs kuriant veiksmingas prevencijos strategijas ir paskirstant sveikatos priežiūros išteklius [2]. Šio populiacinio tyrimo tikslai buvo nustatyti 24 metų laikotarpio GSI sergamumo, mirštamumo ir mirtingumo tendencijas tarp darbingo amžiaus (25–64 metų) Kauno miesto gyventojų 2000–2023 metais ir įvertinti COVID-19 pandemijos įtaką. [...].

Propofol is a widely utilized general anesthetic that facilitates rapid induction and recovery of anesthesia. A major adverse effect of propofol is the reduction in systemic vascular resistance, which frequently results in hypotension. This hemodynamic alteration may precipitate cardiac arrhythmias and myocardial ischemia due to compromised coronary perfusion and diminished oxygen delivery. One principal mechanism underlying propofol-induced hypotension is peripheral vasodilation. Propofol exerts direct relaxant effects on vascular smooth muscle and attenuates sympathetic nervous system activity, thereby decreasing vascular tone. Consequently, systemic vascular resistance declines, leading to a fall in arterial blood pressure. Currently, there is no consistently effective clinical intervention to fully mitigate propofolinduced hypotension, and alternative strategies are often insufficient. The cardiovascular side effects of propofol may involve multiple molecular targets. Previous studies have demonstrated that propofol can modulate intercellular communication via gap junction (GJ) channels composed of connexin 43 (Cx43). Nevertheless, the comprehensive effects of propofol on other vascular connexins remain poorly characterized. Cx45, Cx43, Cx40, and Cx37 are all known to form GJ channels in the vascular system, contributing to vasodilation, synchronization of vascular tone, and the pathogenesis of various vascular disorders, including hypertension and atherosclerosis. The present study aimed to elucidate the effects of propofol on cardiovascular connexins. To this end, we examined the impact of propofol on GJ channels formed by Cx45, Cx43, Cx40, and Cx37, which were exogenously expressed in human cervical epithelial adenocarcinoma (HeLa) cells. Junctional conductance was assessed using the double whole-cell patch-clamp technique. Our results indicated that Cx40 and Cx43 channels exhibited comparable sensitivity to propofol, whereas Cx45 channels responded only at substantially higher drug concentrations. Notably, Cx37 demonstrated the highest susceptibility, displaying significant sensitivity even at low propofol concentrations. Based on these findings, we hypothesized that Cx37 may play a central role in propofol-induced hypotension. Physiologically, Cx37 mediates the transmission of vasodilatory signals, thereby contributing to reductions in vascular resistance. Knockout models have demonstrated that the absence of Cx37 alters vascular responsiveness to diverse stimuli, resulting in endothelial dysfunction. Consequently, we focused on elucidating the mechanistic basis of propofol action on Cx37. Previous reports have suggested that propofol modulates Cx43 GJ channels via activation of protein kinase C (PKC), leading to phosphorylation of Cx43 and reduced intercellular coupling. To determine whether a similar pathway mediates propofol effects on Cx37, we employed the kinase inhibitor GF109203X. Low concentrations of GF109203X selectively inhibit PKC, whereas higher concentrations inhibit both PKC and protein kinase A (PKA). Neither concentration abrogated propofol-induced inhibition of Cx37 channels, indicating that neither PKC nor PKA mediates this effect. To further investigate the underlying mechanism, channel gating experiments were performed using a –90 mV, 45 s ramp protocol. These studies revealed minimal differences between control and propofol conditions, suggesting that propofol mechanism of action is independent of gating alterations. However, further studies are needed to investigate the mechanism of propofol's effect on Cx37.

Connexin-36 (Cx36) forms gap junction (GJ) channels that constitute the majority of electrical synapses in mammalian CNS and enable direct signaling between pancreatic beta cells. GJ channels are formed by the docking of two hexameric Cx hemichannels, each gating in response to the transjunctional voltage, Vj. Two distinct Vj gating mechanisms, attributed to the N-terminal domain (NT) and the first extracellular loop, are operative in each hemichannel and can modulate coupling. Uniquely among the 21 human Cx isoforms, intracellular Mg2+ robustly modulates Cx36 GJs, affecting the magnitude of coupling as well as sensitivity to Vj. Previously, we showed that charge substitutions E3Q, E8Q, A13K, and H18K in NT of Cx36 modified sensitivity to Mg2+. Here, we show that these same charge substitutions also alter Vj dependence. Mathematical modeling indicates that Mg2+ effects alone cannot account for the data, implicating modification of intrinsic Vj gating properties. The NT domain forms the cytoplasmic vestibule of a GJ channel and a number of residues function in sensing Vj and stabilizing open/closed configurations. Molecular dynamics simulations show that each of the NT charge substitutions altered the electrostatic profile of the channel pore and produced widespread alterations in interactions between residues in NT and the transmembrane domains that can affect the stability of the putative open conformation. Using heterotypic pairings of WT Cx36 and variants, we established a positive gating polarity for Cx36 and demonstrated polarity reversal for the E3Q substitution, properties indicative that NT-mediated gating plays a predominant role in Vj-dependence of Cx36 GJs.

This paper presents a review of traditional machine learning algorithms and artificial neural networks for identifying cellular morphological features, subcellular structures, changes in cell division, and viability. It also discusses their applications in predicting and diagnosing various diseases. Finally, it addresses the limitations and challenges that still persist in cell imaging analysis and the restrictions on the broader use of artificial intelligence in medicine.

Unapposed connexin (Cx) hemichannels serve as precursors to gap junction channels but also function independently, playing crucial roles in various physiological processes. Hemichannel gating is influenced by factors such as plasma membrane voltage and extracellular divalent ion concentrations. Excessive hemichannel opening can lead to significant leakage of ions and molecules, and mutations in genes encoding Cxs often result in aberrant gating, contributing to various pathologies. Therefore, evaluating and quantifying Cx hemichannel gating behaviours is important. To address this, we developed a mathematical/computational model describing the voltage-gating properties of Cx hemichannels. The proposed model incorporates two distinct gating mechanisms - fast and loop gating - known to regulate hemichannel closure. These gating transitions are represented within a four-state kinetic scheme, which also accounts for redistribution of voltage upon the closure of either mechanism. Using a sensitivity function matrix approach, we selected voltage protocols that provide sufficient information to constrain the proposed model. The model was then fitted to electrophysiological data recorded from Cx26 and Cx45 hemichannels. Fits to both training datasets and independent validation data indicate that the model can adequately describe the basic characteristics of Cx hemichannel currents. Further analysis using the proposed kinetic scheme provides insights into hemichannel gating behaviour, including the observed delay in current activation upon depolarization and potential discrepancies between gating kinetics of unapposed hemichannels and gap junction channels. Thus, the proposed model can serve as a valuable tool for comparing voltage-gating properties across Cx isoforms and mutants and offers insights into Cx hemichannel gating behaviours. KEY POINTS: Gating of unapposed connexin (Cx) hemichannels plays a crucial role in various physiological processes, whereas mutations in Cx genes that cause aberrant gating are linked to various pathologies. To quantify the voltage-gating properties of Cx hemichannels, we present a novel mathematical/computational model that comprises two established gating mechanisms: fast and loop gating. The validity of the proposed model is demonstrated through fits to electrophysiological data from cells expressing different Cx isoforms, Cx26 and Cx45. The proposed model can serve as a useful tool for comparing the voltage-gating properties across Cx isoforms and mutants, and offers insights into the physiologically relevant mechanistic behaviours of Cx hemichannels.

Gap junction (GJ) channels, formed of connexin (Cx) protein, enable direct intercellular communication in most vertebrate tissues. One of the key biophysical characteristics of these channels is their unitary conductance, which can be affected by mutations in Cx genes and various biochemical factors, such as posttranslational modifications. Due to the unique intercellular configuration of GJ channels, recording single-channel currents is challenging, and precise data on unitary conductances of some Cx isoforms remain limited. In this study, we applied stationary noise analysis, a method successfully used for ion channels with very low unitary conductances, to GJ channels. We modified this technique to account for the residual conductance of GJ channels and present three strategies for estimating unitary conductance, including model-based evaluation of open-state probability and subtraction of residual conductance. To assess the validity, advantages, and limitations of these approaches, we performed mathematical analysis and simulation experiments. We also addressed practical issues such as the underestimation of sample variance in autocorrelated recordings and channel rundown, proposing solutions to these issues. Finally, we applied these strategies to electrophysiological data recorded from cells expressing Cx45. Our findings showed that noise-based estimates of Cx45 unitary conductance from macroscopic currents align well with those obtained from single-channel recordings.

Propofol is a widely used general anesthetic, which causes a rapid induction of anesthesia. The most prominent side effect of propofol is the decrease of systemic vascular resistance that leads to hypotension. Moreover, in some cases, propofol has been shown to inhibit cardiac conduction and cause bradycardia, but its pathophysiological mechanism is still not fully understood. On the other hand, propofol was shown to have a protective effect against ventricular arrhythmias during myocardial ischemia. Several studies have shown that propofol may regulate cell coupling through gap junction (GJ) channels formed of Cx43. However, it is unclear how propofol affects all cardiac connexins. It is known that Cx37, Cx40, Cx43 and Cx45 form gap junction channels in cardiovascular system. These connexins are required for coordination of vascular responses and play a key role in ensuring propagation of action potential in cardiac tissue. The aim of this study was to get a better understanding of the capacity of propofol to affect cardiovascular connexins. First, we compared the effect of various propofol concentrations on GJs formed by cardiovascular connexins which were expressed exogenously in human cervix epithelial adenocarcinoma cells (HeLa). The junctional conductance was measured using double whole-cell patch clamp method. Our data show that Cx40 and Cx43 channels exhibit similar sensitivity to propofol (IC50 Cx40 - 17µM; Cx43 – 15µM), while Cx45 channels are sensitive to much higher propofol concentrations (IC50 60µM). Interestingly, the vascular Cx37, which is an important player in dilation of vascular beds, was the most susceptible to propofol (IC50 5µM). Propofol may affect Cx43 GJ channels through activation of protein kinase C (PKC), which in turn phosphorylates Cx43 and reduces coupling through GJs. In our study, the kinase inhibitor GF109203X was used to assess this putative pathway of propofol action on GJs. It is established that low concentrations (40nM) of GF109203X specifically inhibit PKC while higher concentrations (2 µM) inhibit both PKC and protein kinase A (PKA). Our data showed that low concentration of GF109203X significantly reduced inhibition of Cx43 channels by propofol. In contrast, both concentrations of GF109203X had no effect on inhibition of Cx40 and Cx45 channels by propofol. The findings indicate that both low and high kinase inhibitor concentrations considerably lessened propofol’s blocking effect on Cx37.