Biology subjects

Nikolaev, V. O.

Publications and source records attributed to Nikolaev, V. O..

3 recordsLinked to original sources

bioRxiv2025

Differential contribution of HCN1 and HCN4 to the synchronisation of sinoatrial pacemaker cells

The hearts ability to beat with high precision, with a regular and steady rhythm relies on the synchronised activity of pacemaker cells in the sinoatrial node (SAN), which communicate with one another through gap junctions. This process ensures that electrical impulses are organised and reach a critical mass to ignite the electrical activity of the surrounding atrial tissue and trigger the regular heartbeat. HypothesisWe hypothesise that hyperpolarisation-activated, cyclic nucleotide-gated (HCN) channels play a pivotal role in maintaining this synchronisation process. This idea aligns with the well-established role of HCN channels in stabilising the membrane potential in the voltage range of the slow diastolic depolarisation, counteracting voltage fluctuations and effectively filtering out variations in the beating rate from neighbouring cells. AimWe focus on two specific HCN channel subtypes--HCN1 and HCN4--and their contributions to the rapid synchronisation of pacemaker cells, a phenomenon known as phasic entrainment. Using two HCN channel-mutant mouse models, we dissect the distinct roles of HCN1 and HCN4 in this process. MethodsWe employed patch-clamp electrophysiology to determine phase response curves (PRCs) to predict the ability of single cells to interact in the SAN. Using computer simulations, the behaviour of the SAN at the network level was determined. ResultsWe found that HCN1, but not HCN4, is essential for the fast synchronisation of pacemaker cells in the SAN and propose a mechanism by which HCN1 channels regulate this process. ConclusionThese findings highlight HCN1 as a critical component for ensuring the precise and rapid coordination needed for synchronisation of the SAN, for a regular and consistent heartbeat. Translational perspectiveOur work provides essential insights into the cellular and molecular mechanisms governing SAN function and lays the groundwork for several clinically relevant applications. Understanding how If blockers may affect heart rate and rhythm stability is crucial for assessing potential side effects of current and future subtype-specific HCN channel inhibitors. Moreover, our findings could support new diagnostic strategies for detecting patients at risk of SAN dysfunction. Ultimately, these findings pave the way for innovative therapeutic approaches, including targeted channel modulation and future cell or gene therapies to restore pacemaker stability.

physiology↗

bioRxiv2025

RBM20-variants induce distinct calcium handling and metabolic phenotypes in patient-specific stem cell models of dilated and non-compaction cardiomyopathy

Background and aimMutations in the splice regulator RBM20 account for [~]3 % of genetic cardiomyopathies. In particular, the highly conserved RS domain is a hotspot for disease-associated mutations. Previously, mutations at same amino acid position 634 in the hotspot RS-domain were found to cause dilated cardiomyopathy (DCM) with left ventricular non-compaction (R634L) or without (R634W), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remained unknown. Here, we identify the molecular events caused by the distinct RBM20 mutations from DCM and left-ventricular non-compaction (LVNC) using patient-specific stem cell models. MethodsWe generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) of one LVNC- and two DCM-patients harboring the RBM20-mutations R634L (LVNC) or R634W (DCM). We investigated alternative splicing activity, RBM20 localization, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of key Ca2+ handling enzymes, physiological cardiac functions as Ca2+ homeostasis, and metabolic activity on a patient-specific cardiomyocyte level. Force generation was analyzed in patient-specific engineered myocardial tissues. Isogenic rescue and mutation insertion lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations on the cardiac phenotype. ResultsWe observed common splicing aberrations for LVNC- and DCM-CM in TTN and RYR2, RBM20 cytoplasmatic accumulation and irregular sarcomeric structure. LVNC-CM harboring the RBM20-p.R634L variant show distinct molecular, cellular and functional impairments that manifest in CAMK2D, TRDN and IMMT mis-splicing. Splicing defects in LVNC-CM correlate with elevated systolic Ca2+ and faster Ca2+ kinetics with elevated cAMP levels and PLN-hyperphosphorylation. An increased metabolic activity and mitochondrial membrane potential support the hyperactive LVNC-CM. By contrast, DCM-CM (RBM20-p.R634W) distinctly present with decreased systolic Ca2+ and increased SR Ca2+leak but unchanged Ca2+ kinetics and metabolic activity. Both mutations lead to severely reduced force of contraction in engineered myocardium. CRISPR/Cas9 gene-edited isogenic control lines of both described RBM20 mutations in LVNC and DCM demonstrated the causative nature of the two mutations and their diverging effects. Further, L-type Ca2+ channel blockade by verapamil ameliorates the Ca2+ cycling and leakage phenotypes in LVNC- and DCM-CM. ConclusionWe show the first comparative iPSC-model of splice-defect-associated RBM20-dependent LVNC-p.R634L and DCM-p.R634W. We found shared and variant-specific phenotypes on a patient-specific level. Our data demonstrate that the different RBM20 mutations manifest in distinct molecular aberrations in alternative splicing and RBM20 cytoplasmic accumulation that convey various physiological impairments in structure, Ca2+ handling, metabolism and contractile force.

molecular biology↗

bioRxiv2019

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles

Summary sentenceBoth FSH and LH increase Ca2+ in the granulosa cells of intact ovarian follicles from mice expressing genetically encoded sensors.\n\nABSTRACTIn mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at ~10 minutes, remains above baseline for another ~10 minutes, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 hours and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does slightly attenuate ovulation. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.

cell biology↗