Biology subjects

Kohl, P.

Publications and source records attributed to Kohl, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Channelrhodopsin Ion Selectivity Determines Mechanisms and Efficacy of Optogenetic Defibrillation in Human Atria and Ventricles

Optogenetic defibrillation uses light-gated ion channels to terminate cardiac arrhythmias through targeted illumination. Previous studies assessed the feasibility of using either cation (e.g. ChR2) or anion (e.g. GtACR1) non-selective channels, both of which depolarise resting cardiomyocytes upon photoactivation. In contrast, recently identified light-gated K+-channels (e.g. WiChR) suppress cardiomyocyte activity while maintaining the membrane potential near its resting state. Here, we use biophysically detailed simulations to compare the defibrillation potential of ChR2, GtACR1, and WiChR. Single-cell simulations show that activation of ChR2 and GtACR1 markedly increase diastolic intracellular Ca2+ concentration (by 42.6% and 52.6%, respectively), whereas WiChR induces only minimal changes (4.0% increase), suggesting a lower pro-arrhythmogenic risk. WiChR activation, however, slightly increases intracellular Na+ levels (by 15.1% compared to 0.1% and 3.4% for ChR2 and GtACR), consistent with the residual Na+ permeability of all currently available K+-selective channelrhodopsins. Simulations of human ventricles and atria demonstrate that GtACR1 most effectively terminates re-entrant arrhythmias at low light intensities, while WiChR achieves comparable efficacy at light levels [≥]5 mW/mm2. Complementary tissue-scale simulations reveal that defibrillation is either based on depolarisation within the excitable gap, followed by fast Na+ channel inactivation (depolarising variants ChR2 and GtACR1), or based on a reduction in membrane resistance supporting arrhythmia termination at sufficiently high light levels (large-conductance ion channels GtACR1 and WiChR). Overall, our findings identify channelrhodopsin ion selectivity as a key determinant of both arrhythmia termination success and mechanisms underlying defibrillation. Key points summaryO_LIWe use computational simulations to compare non-selective cation (ChR2), anion (GtACR1), and K+-selective channelrhodopsins (WiChR) for optogenetic termination of re-entrant arrhythmia. C_LIO_LISingle-cardiomyocyte simulations suggest that ChR2 and GtACR1 activation can cause progressive accumulation of intracellular Ca2+, which is minimised when using WiChR. C_LIO_LISimulations of human left ventricles and atria indicate that GtACR1 is most effective in terminating re-entrant arrhythmia at low light intensities, while WiChR becomes similarly effective at higher intensities. C_LIO_LITissue-scale simulations indicate distinct defibrillation mechanisms: Excitable gap extinction by de-novo action potential initiation followed by inactivation of fast Na+ channels for depolarising channelrhodopsins (ChR2, GtACR1), and reduction in membrane resistance for the large-conductance channels (GtACR1, WiChR), effectively clamping the membrane potential at each channels reversal potential at high light levels. C_LI

physiology↗

bioRxiv2026

Cross-species architecture of the cardiac transverse-axial tubular system in mammals

Cardiac excitation-contraction coupling relies on a pancellular network of regular cardiomyocyte surface membrane invaginations, termed the transverse-axial tubular system (TATS). The TATS is ubiquitously present in adult mammalian cardiomyocytes, enabling efficient structural and functional coupling of sarcolemma and intracellular Ca2+ stores. However, TATS ultrastructural characteristics across species, and their relation to cardiomyocyte morphology and physiological parameters such as heart rate, remain largely unexplored. Here, we quantified TATS and cardiomyocyte features in a large confocal microscopy dataset (78 3D volumes) obtained from tissue slices across eight species (mouse, rat, rabbit, pig, horse, elephant, whale, and human). We developed and applied a semi-automated image analysis pipeline to quantify mean cytosolic distances to the nearest TATS (Cyto-TATSmin, a measure inversely related to TATS density), transverse-to-axial tubule ratio, and cardiomyocyte dimensions. Cyto-TATSmin and transverse tubule fraction differed substantially between species, with the lowest Cyto-TATSmin in mouse and highest in human. Within species, except mouse, rat, and horse, Cyto-TATSmin was positively correlated with cardiomyocyte cross-sectional area. Across all species, Cyto-TATSmin correlated with species life span and body weight, and was inversely correlated with average resting heart rate. Our findings reveal structural scaling principles within species differences in cardiac cellular ultrastructure and provide a resource for studying TATS organisation in health and disease. As TATS remodelling is a common hallmark of cardiac pathology, awareness of species-differences is needed to guide the design and interpretation of translational research.

cell biology↗

bioRxiv2026

Cardiomyocyte mechanical activity counteracts intraluminal calcium depletion in the transverse-axial tubular system during fast electrical stimulation

The transverse-axial tubular system (TATS) enables close structural and functional coupling between plasma membrane and sarcoplasmic reticulum of cardiomyocytes. It supports fast and efficient Ca2+-induced Ca2+ release upon cell depolarisation, crucial for excitation-contraction coupling in the heart. Due to the small diameter and tortuosity of individual tubules, the TATS forms a domain of restricted diffusive transport. It has previously been suggested that, as a consequence of an uneven distribution of Ca2+ influx and efflux pathways in TATS compared to outer surface plasma membrane domains of cardiomyocytes, cyclic electrical activity may lead to a gradual depletion of Ca2+ in the TATS. Here, we show experimentally that in mechanically uncoupled rabbit ventricular cardiomyocytes, electrical stimulation does indeed lead to an L-type Ca2+ channel-dependent gradual depletion of Ca2+ inside TATS, an effect that scales with pacing frequency. Ca2+ depletion was absent in freely contracting cardiomyocytes, presumably as a result of cyclic TATS deformation during cell shortening. This squeezes transverse TATS tubules and adds an advective contribution to, and thereby accelerates the, intra-TATS content exchange with bulk extracellular fluid. Our results reveal a novel mechanism of cardiac mechano-dependent auto-regulation, where the increased propensity for development of intra-TATS Ca2+ gradients at high electrical stimulation rates is mitigated by the coinciding mechanically induced TATS deformation, twice on each cycle in the heart (during diastolic stretch and systolic shortening), which accelerates luminal content exchange. Our study provides first insight into a novel facet of cardiac mechano-biology, whose auto-regulatory benefit may be reduced by TATS remodelling in disease.

cell biology↗

bioRxiv2024

Disturbed repolarisation-relaxation coupling during acute ischaemia permits systolic mechano-arrhythmogenesis.

BackgroundThe hearts mechanical state feeds back to its electrical activity, potentially contributing to arrhythmias. Mechano-arrhythmogenesis has been mechanistically explained during electrical diastole, when cardiomyocytes are at their resting membrane potential. During electrical systole, cardiomyocytes are refractory right from the onset of depolarisation, while during repolarisation they appear to be protected from mechano-arrhythmogenesis by near-simultaneous restoration of resting membrane potential and cytosolic calcium concentration ([Ca2+]i): repolarisation-relaxation coupling (RRC). Yet, systolic mechano-arrhythmogenesis has been reported in ischaemic myocardium, with unclear underlying mechanisms. We hypothesise that ischaemia-induced alteration of RRC gives rise to a vulnerable period for mechano-arrhythmogenesis. MethodsAcute left-ventricular (LV) regional ischaemia was induced by coronary artery ligation in Langendorff-perfused rabbit hearts, with mechanical load controlled by an intraventricular balloon. Mechanical activity was assessed by echocardiography and arrhythmia incidence by electrocardiogram. Single LV cardiomyocytes were exposed to simulated ischaemia or pinacidil (ATP-sensitive potassium channel opener). Stretch was applied in diastole or late systole using carbon fibres. Stretch characteristics and arrhythmia incidence were assessed by sarcomere length measurement. In both models, RRC was assessed by simultaneous voltage-[Ca2+]i fluorescence imaging and mechano-arrhythmogenesis mechanisms were pharmacologically tested. ResultsIn whole heart, acute regional ischaemia leads to systolic stretch and disturbed RRC at the ischaemic border. These electro-mechanical changes were associated with waves of arrhythmias, which were reduced by mechanical unloading, electro-mechanical uncoupling, or buffering of [Ca2+]i. In LV cardiomyocytes, physiological RRC is associated with a low incidence of systolic mechano-arrhythmogenesis, while a vulnerable period emerged by prolonged RRC during ischaemia. The increase in systolic mechano-arrhythmogenesis was reduced by restoring RRC, chelating [Ca2+]i, blocking mechano-sensitive transient receptor potential kinase ankyrin 1 channels (TRPA1), or buffering reactive oxygen species (ROS) levels. ConclusionProlonged RRC allows for systolic mechano-arrhythmogenesis in acute ischaemia, involving contributions of elevated [Ca2+]i, TRPA1 activity, and ROS, which represent potential anti-arrhythmic targets. GRAPHICAL ABSTRACT LEGENDRole of disturbed repolarisation-relaxation coupling (RRC), transient receptor potential kinase ankyrin 1 (TRPA1) channels, cytosolic calcium concentration ([Ca2+]i), and reactive oxygen species (ROS) in ventricular systolic mechano-arrhythmogenesis. Schematic of the proposed mechanisms underlying the TRPA1- and Ca2+-mediated increase in systolic mechano-arrhythmogenesis with disturbed RRC. AITC, Allyl isothiocyanate (TRPA1 channel activator); AP, action potential; BAPTA ([Ca2+]i buffer); CaT, Ca2+ transient; DNT, dantrolene (ryanodine receptor stabiliser); DPI, diphenyleneiodonium (ROS production blocker); GLIB, glibenclamide (KATP channel blocker); HC-300031 (TRPA1 channel blocker); KATP, ATP-sensitive potassium channel; NAC, N-acetyl-L-cysteine (ROS chelator); NCX, sodium-Ca2+ exchanger; PIN, pinacidil (KATP channel activator); ROS, reactive oxygen species; SI, simulated ischaemia; STP, streptomycin (non-specific mechano-sensitive ion channel blocker).

physiology↗

bioRxiv2024

Spatio-temporal dynamics of the fibrotic niche in cardiac repair

The heart is one of the least regenerative organs in humans, and ischemic heart disease is the leading cause of death worldwide. Understanding the cellular and molecular processes that occur during cardiac wound healing is an essential prerequisite to reducing health burden and improve cardiac function after myocardial tissue damage. By integrating single-cell RNA-sequencing with imaging-based spatial transcriptomics, we reconstructed the spatio-temporal dynamics of the fibrotic niche after ventricular injury in adult mice. Our analysis reveals dynamic regulation of local cell communication niches over time. We identified interactions that regulate cardiac repair, including fibroblast proliferation silencing by Trem2high macrophages that prevents excessive fibrosis. Moreover, we discovered a rare population of dedifferentiating cardiomyocytes during early post-lesion stages, which was sustained by signals from myeloid and lymphoid cells. Culturing non-regenerative mouse cardiomyocytes or human heart tissue with these niche factors reactivated progenitor gene expression and cell cycle activity. In summary, this spatio-temporal cell type atlas provides valuable insights into the heterocellular interactions that control cardiac repair. HighlightsO_LIscRNA-seq and in situ sequencing reveal spatio-temporal dynamics of heart repair C_LIO_LILocal heterocellular communication niches coordinate overall wound response C_LIO_LIFibroblast cell cycle silencing by Trem2high macrophages suppresses excessive fibrosis C_LIO_LICardiomyocyte plasticity is promoted by myeloid and lymphoid cells C_LI

systems biology↗

bioRxiv2024

Repolarisation-relaxation dyscoupling and TRPA1 activation permit systolic mechano-arrhythmogenesis

BackgroundThe hearts mechanical state feeds back to its electrical activity, potentially contributing to arrhythmias (mechano-arrhythmogenesis, MAR). MAR has been mechanistically explained during electrical diastole, when cardiomyocytes are at their resting membrane potential. Conversely, during electrical systole, cardiomyocytes appear to be protected from MAR, even as membrane potential and cytosolic calcium concentration ([Ca2+]i) are simultaneously restored to resting levels during repolarisation (repolarisation-relaxation coupling, RRC). Yet, systolic MAR has been reported in ischaemic myocardium, with unclear underlying mechanisms. MethodsRabbit left ventricular cardiomyocytes were electrically paced and exposed to a simulated ischaemia solution (including hyperkalaemia, acidosis, and block of oxidative phosphorylation) or pinacidil (to simulate ischaemia-induced opening of ATP-sensitive potassium [KATP] channels), with or without glibenclamide (to block KATP channels). RRC was assessed by simultaneous measurement of membrane voltage and [Ca2+]i dynamics with fluorescence imaging. Acute stretch at increasing magnitudes was applied using carbon fibres, with stretch timed to diastole or late systole. Stretch mechanics and the incidence of MAR was assessed by video-based measurement of sarcomere length. Mechanisms contributing to MAR were assessed by buffering [Ca2+]i (BAPTA-AM), stabilising ryanodine receptors (dantrolene), non-specifically blocking mechano-sensitive channels (streptomycin), activating (AITC) or blocking (HC-030031) transient receptor potential kinase ankyrin 1 channels (TRPA1), or chelating (NAC) or blocking production of (DPI) reactive oxygen species (ROS). ResultsIt was reconfirmed that MAR during physiological RRC is rare, while ischaemia- or pharmacologically-induced RRC dyscoupling generates a vulnerable period for systolic MAR. This systolic MAR depends on TRPA1, [Ca2+]i, and ROS, which contribute to stretch-induced excitation and arrhythmia sustenance. An increase in systolic MAR can be prevented by mitigating RRC dyscoupling with KATP channel block, or by blocking TRPA1, buffering [Ca2+]i, or reducing ROS. ConclusionRRC dyscoupling may be arrhythmogenic in ischaemia and other pathologies associated with systolic MAR, and TRPA1 may be a novel anti-arrhythmic target.

physiology↗

bioRxiv2023

3D structure of fibroblasts and macrophages in the healthy and cryo-ablated heart

IntroductionCardiac non-myocytes (NM) play important roles in heart development, homeostasis, and lesion repair. To assess the relevance of different NM populations for cardiac (patho)physiology, a quantitative assessment of their abundance and structure in the different heart chambers is an essential prerequisite. We here present an experimental approach to determine the distribution, dimensions, and 3D morphology of fibroblasts (FB) and macrophages (M{Phi}) in healthy and pathologically remodelled hearts. Methods and resultsWe used Cre-loxP recombination to selectively target channelrhopsin-2 (ChR2)-eYFP to either FB or M{Phi} in healthy and cryo-ablated mouse hearts. Hearts were optically cleared using X-CLARITY and membrane-bound eYFP fluorescence was recorded by confocal microscopy. The resulting image stacks were segmented to generate 3D reconstructions of labelled cell populations in near native tissue. In doing so, we show that FB and M{Phi} have similar surface areas, volumes and morphologies, but that FB occupy larger fractional volumes than M{Phi} in all chambers of healthy murine hearts. Furthermore, M{Phi} appear primarily as single cells, whereas FB form extended networks of interconnected cells. In left-ventricular tissue following cryo-ablation, we observed large disordered networks of FB in the scar area with an increased volume occupied by FB both in the scar and remotely. In cryo-ablated ventricles, M{Phi} form comparatively small, but dense networks in the scar without changing their abundance in remote myocardium. ConclusionsOur study assesses the 3D distribution and structure of fluorescently labelled FB and M{Phi} in healthy and lesioned murine hearts. Based on 3D reconstructions of FB and M{Phi} networks, we quantified the surface areas and volumes of individual non-myocytes in the different chambers of the heart and in ventricular scar tissue, thus providing important quantitative data serving as basis for computational modelling of non-myocyte contributions to cardiac structure and physiology.

cell biology↗

bioRxiv2023

Perivascular Excitation Tunnelling: a Novel and Preventable Cause of Cardiac Reperfusion Arrhythmias

BackgroundReperfusion after myocardial ischaemia can lead to deadly arrhythmias, in part due to heterogeneities in electrophysiology (EP) across affected tissue. There is a need to understand the spatiotemporal dynamics of ischaemia-reperfusion arrhythmias (IRA), so that reperfusion strategies to prevent them can be found. MethodsLangendorff-perfused rabbit isolated hearts were loaded with a voltage-sensitive dye. Epifluorescence imaging was used to track action potential propagation across the cardiac surface. The heart was simultaneously perfused globally (via the aorta) and locally (via cannulation of a single coronary artery) with an oxygenated physiological saline solution. Local perfusion was subsequently switched to and from solutions that mimic aspects of ischaemia (acidosis, hypoxia, hyperkalaemia, or a simulated ischaemia solution combining all three) or to no-flow. Subsequently, different reperfusion strategies were tested to reduce IRA re-entries. The most successful strategy for preventing re-entry was tested in Langendorff-perfused isolated pig hearts to assess the clinical relevance of the observed mechanism and treatment strategy. ResultsUpon sudden reperfusion of the cannulated coronary artery in rabbit hearts we observed a preferential recovery of electrical excitability along the vessels main branch ( perivascular excitation tunnelling, PVET). This resulted in re-entry in roughly half of the hearts. Hyperkalaemia and hypoxia, but not acidosis, were sufficient to lead to conduction block, PVET, and re-entry, with both PVET and re-entry more frequently observed after hyperkalaemia than hypoxia. PVET was also present in pigs and PVET-based re-entries were successfully prevented in rabbit and pig hearts by two-step reperfusion, first of the distal majority of the previously ischaemic region, and then of the remaining tissue from the proximal point. With this strategy, any PVET that developed in the distal tissue was blocked by the still inexcitable proximal tissue. Upon reperfusion of the proximal tissue, there was a reduced path length for PVET. As a consequence, the associated excitable gap was too short for re-entrant excitation. ConclusionsWe observed a novel arrhythmia mechanism upon coronary reperfusion (PVET), which suggests that preferential recovery of myocardial excitability along the reperfused vessel is an important mechanism underlying IRA formation. PVET-induced re-entry reliably occurred in both rabbit and pig hearts and could be prevented by two-step reperfusion. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/569031v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@139a095org.highwire.dtl.DTLVardef@5dc811org.highwire.dtl.DTLVardef@f9db75org.highwire.dtl.DTLVardef@1b6f66b_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-coordinated excitation-contraction coupling (ECC) and, thus, homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarisation is active (local generation of action potentials; AP) or passive (following depolarisation of the outer cell surface sarcolemma; SS) has not been experimentally assessed in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesise that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were electrically insulated from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTSO_LICardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, ensuring synchronous and uniform cell contraction. C_LIO_LIIn this study, we investigate whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. C_LIO_LIFor this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of sub-cellular membrane domains. C_LIO_LIOur findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. C_LIO_LIThis active electrical capability may serve as a protective mechanism against voltage drops occurring within the T-tubular network. C_LI

physiology↗

bioRxiv2023

Structure and dynamics of human cardiac fibroblast nanotubes

Efficient and dynamic interactions between cardiac fibroblasts and their environment are essential for the maintenance of tissue homeostasis in healthy hearts and play an important role during pathological remodelling. Here, we investigate a relatively obscure mechanism through which human atrial fibroblasts communicate with each other, with other cells, and with the extracellular matrix (ECM) - nanotubes (NT). We investigated NT structure and dynamics in primary right atrial fibroblasts isolated from patients in sinus rhythm (SR) and atrial fibrillation (AF), in an immortalised human atrial fibroblasts cell line, and in intact human tissue, using a wide range of imaging approaches (including confocal microscopy, label-free reflection microscopy, rotating coherent scattering microscopy, and cryo-electron tomography). We show that fibroblasts maintain continuous NT activity in vitro, with numerous protrusions constantly probing the surrounding environment. NT structure and activity change during AF and following pharmacological (transforming growth factor-{beta}, latrunculin B) and environmental (hypoxia) interventions. We also show that cardiac fibroblast NT mediate intercellular organelle exchange and dynamically interact with ECM. Finally, we present evidence for the presence of fibroblast-borne NT in human atrial tissue. Our results advance our understanding of how cardiac fibroblasts interact with their environment. NT are versatile structures capable of both sensory and actuating functions, and offer a dynamic and rapid communication conduit that facilitates cell-cell and cell-extracellular matrix interactions.

cell biology↗

bioRxiv2023

Piezo1 stretch-activated channel activity differs between bone marrow-derived and cardiac tissue-resident macrophages

Macrophages (M{Phi}) play pivotal roles in tissue homeostasis and repair. Their mechanical environment recently emerged as a key modulator of various cell functions, and M{Phi} mechanosensitivity is likely to be critical for cellular activity in particular in a rhythmically contracting organ such as the heart. M{Phi}, in-vitro-differentiated from bone marrow (M{Phi}BM), form a popular cell model for research. This study explores the activity of stretch-activated ion channels (SAC) in murine M{Phi}BM and compares it to SAC activity in cardiac tissue-resident M{Phi} (M{Phi}TR). Our main findings are: i) M{Phi}BM and M{Phi}TR have stretch-induced currents, indicating expression of functional SAC at their plasma membrane; ii) the current profiles in M{Phi}BM and in M{Phi}TR show characteristics of cation non-selective SAC; iii) unlike in M{Phi}BM, Piezo1 ion channel activity at the plasma membrane of M{Phi}TR is not detectable, neither by assessing electrophysiological activity using the patch clamp technique, nor by measuring cytosolic calcium concentration upon perfusion with Yoda1, a Piezo1 channel agonist. In mature scars after ventricular cryoablation, stretch-induced current characteristics of M{Phi}TR are not significantly different compared to non-injured control tissue, even though scars are expected to contain a mix of pre-existing and circulation-recruited M{Phi}. This suggests that M{Phi} invading injured cardiac tissue either phenoconvert their mechanosensitivity from M{Phi}BM to M{Phi}TR, or that the in vitro differentiation protocols used to obtain M{Phi}BM generate cells that differ from M{Phi} recruited from the circulation during tissue repair in vivo. Further investigations will explore SAC identity in lineage-traced M{Phi} in scar tissue, and compare mechanosensitivity of circulating monocytes with that of M{Phi}BM. Key pointsO_LIM{Phi}BM and M{Phi}TR have stretch-induced currents, indicating expression of functional SAC at their plasma membrane; C_LIO_LIThe current profiles in M{Phi}BM and in M{Phi}TR show characteristics of cation non-selective SAC; C_LIO_LIUnlike in M{Phi}BM, Piezo1 ion channel activity at the plasma membrane of M{Phi}TR is not detectable C_LI

biophysics↗

bioRxiv2023

Age-related structural and functional changes of the intracardiac nervous system

BackgroundAlthough aging is known to be associated with an increased incidence of both atrial and ventricular arrhythmias, there is limited knowledge about how Schwann cells (SC) and the intracardiac nervous system (iCNS) remodel with age. Here we investigate the differences in cardiac SC, parasympathetic nerve fibers, and muscarinic acetylcholine receptor M2 (M2R) expression in young and old mice. Additionally, we examine age-related changes in cardiac responses to sympathomimetic and parasympathomimetic drugs. Methods and ResultsLower SC density, lower SC proliferation and fewer parasympathetic nerve fibers were observed in cardiac and, as a control sciatic nerves from old (20-24 months) compared to young mice (2-3 months). In old mice, CSPG4 was increased in sciatic but not cardiac nerves. Expression of M2R was lower in ventricular myocardium and ventricular conduction system from old mice compared to young mice, while no significant difference was seen in M2R expression in sino-atrial or atrio-ventricular node pacemaker tissue. Heart rate was slower and PQ intervals were longer in Langendorff-perfused hearts from old mice. Ventricular tachycardia and fibrillation were more frequently observed in response to carbachol administration in hearts from old mice versus those from young mice. ConclusionsOn the background of reduced presence of SC and parasympathetic nerve fibers, and of lower M2R expression in ventricular cardiomyocytes and conduction system of aged hearts, the propensity of ventricular arrhythmogenesis upon parasympathomimetic drug application is increased. Whether this is caused by an increase in heterogeneity of iCNS structure and function remains to be elucidated. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/568538v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11dbccdorg.highwire.dtl.DTLVardef@1563d75org.highwire.dtl.DTLVardef@dcda58org.highwire.dtl.DTLVardef@182e598_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

TREK-1 knock-down in human atrial fibroblasts leads to a myofibroblastic phenotype: a role in phenoconversion and overview of mechano-sensitive channel mRNA expression in cardiac diseases.

Cardiac cell mechanical environment changes on a beat-by-beat basis and also in the course of various cardiac diseases. Cells sense and adapt to such mechanical cues via specialized mechano-sensors mediating adaptive signaling cascades. Here, we report TREK-1 mRNA expression and activity in human atrial fibroblasts and reveal a cross talk between TREK-1 and fibroblast phenoconversion. With the aim to reveal additional candidates underlying mechano-transduction relevant to cardiac diseases, we investigated mechano-sensitive ion channels (MSC) mRNA expression in diseased and non-diseased human hearts. Our results showed higher TREK-1 expression and activity in fibroblasts compared to myofibroblasts and we found that TREK-1 down-regulation leads a more myofibroblastic phenotype suggesting a role for this mechano-sensor in phenoconversion. In addition, TREK-1 is preferentially expressed in the left atrium compared to the right one and its expression is not significantly changed when fibroblasts from patients in sinus rhythm vs. sustained atrial fibrillation are compared. At the whole-heart level, numerous MSC were differentially expressed between atrial and ventricular or between non-diseased and diseased tissue samples. Thus, we identify atrial fibroblast-specific TREK-1 expression and activity and reveal a role of TREK-1 in atrial fibroblast pheno-conversion. We also provide a comprehensive overview of cardiac MSC mRNA expression in atrial and ventricular tissue from diseased and non-diseased patients, identifying potential novel candidates underlying mechanotransduction in cardiac diseases.

cell biology↗

bioRxiv2022

Complexes of tubulin oligomers and tau form an intervening network cross-bridging microtubules into bundles

The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) that are implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. While the role of tau in MT bundling is poorly understood, tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in response to varying concentrations of divalent cations (Mg2+ or Ca2+) in dissipative reaction mixtures containing {beta}-tubulin, full-length tau, and GTP at 37{degrees}C. A concentration-time kinetic phase diagram generated by synchrotron small-angle X-ray scattering (SAXS) reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. Remarkably, SAXS analysis combined with TEM of plastic embedded samples provides direct evidence of an intervening network (IN) of complexes of tubulin oligomers and tau ({approx}5 nm wide filaments), which stabilize MT bundles. In this model, {beta}-tubulin oligomers in the IN are crosslinked by taus MT binding repeats, which also link {beta}-tubulin oligomers to {beta}-tubulin within the MT lattice. The finding of a new role for tubulin revises current dogma where cross-bridging of MTs is attributed entirely to interactions between MAPs. The tubulin-tau complexes of the IN should enhance the barrier properties of MT fascicles in preventing tau missorting to the somatodendritic compartment as happens during neurodegeneration. Furthermore, tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau promoting nucleation and growth of tau fibrils in tauopathies. Significance StatementA cell free model of microtubule (MT) bundles of the axon-initial-segment (known as MT fascicles) was studied in physiologically relevant buffer conditions. MT fascicles have a role in retaining neuronal protein tau, a key protein stabilizing MTs, in the axon. X-ray scattering and electron microscopy led to the discovery of complexes of tubulin oligomers and tau as building blocks of an intervening network that cross-bridge MTs into stable bundles with precisely the same linear geometry observed in-vivo in neurons. Significantly, changes to the chemical structure of tau because of abnormal interactions with cellular enzymes, would be predicted to disrupt the intervening tubulin-tau network and the MT-fascicles barrier function, promoting leakage of tau to the somatodendritic compartment and neuron degradation.

biophysics↗

bioRxiv2022

A Genome-Scale Metabolic Model for the Smut-Fungus Ustilago maydis

Ustilago maydis is an important plant pathogen causing corn-smut disease and an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of environmental adaptation and a full use of the organisms metabolic potential. Here, we report the first genome scale metabolic model (GSMM) of Ustilago maydis (iUma22) for the simulation of metabolic activities. iUma22 was reconstructed from sequencing and annotation using PathwayTools, the biomass equation was derived from literature values and from the codon composition. The final model contains over 25% of annotated genes in the sequenced genome. Substrate utilization was corrected by Biolog-Phenotype arrays and exponential batch cultivations were used to test growth predictions. A pan-genome of four different U. maydis strains revealed missing metabolic pathways in iUma22. The majority of metabolic differences between iUma22 and the pangenome occurs in the inositol, purine and starch metabolic pathways. The new model allows studies of metabolic adaptations to different environmental niches as well as for biotechnological applications.

systems biology↗

bioRxiv2021

Protein Charge Neutralization is the Proximate Driver Dynamically Tuning a Nanoscale Bragg Reflector

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that reduction in repulsive net charge of the purified, recombinant reflectin - either (for the first time) by generalized anionic screening with salt, or by pH titration - drives a finely tuned, precisely calibrated increase in size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the sphericity, size and low polydispersity of the assemblies. Precise proportionality between assembly size and charge-neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth. The resulting stability of reflectin assemblies with time ensures reciprocally precise control of the particle number concentration, thereby encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The results presented here strongly suggest that it is charge neutralization, rather than any change in aromatic content, that is the proximate driver of assembly, fine-tuning a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

molecular biology↗

bioRxiv2021

Transcriptome profile of the zebrafish atrioventricular canal reveals molecular signatures of pacemaker and valve mesenchyme

The atrioventricular canal (AVC) is an essential feature of the heart, which separates the atrium from the ventricle. During heart morphogenesis, it is a hub of molecular processes necessary for distinguishing heart regions; most importantly, for the formation of the AV conduction system and cardiac valves. To better understand the molecular processes underlying AVC development and function, we utilized the transgenic zebrafish line sqet31Et with EGFP expression in the AVC region to isolate this cell population by FACS and profiled its transcriptome by RNA-seq at 48 and 72 hours post fertilization (hpf). Compared to the rest of the heart, the AVC is enriched for the expression of molecular markers associated with mammalian AVC and AV node, including cx36.7 and cx45 which encode connexins forming low conductance gap junctions. Using the transgenic line Tg(myl7:mermaid) encoding the voltage-sensitive fluorescent protein, we showed that loss of function of Isl1 abolished the pacemaker-containing sinoatrial ring (SAR) and resulted in an erratic spread of excitation pattern from the SAR to AVC, indicating the dysfunction of the primary pacemaker. Concurrently, ectopic excitation in the AVC region was observed, suggesting that the zebrafish AVC possesses inherent automaticity although insufficient to replace the primary pacemaking activity of the SAR. Comparisons between the SAR and AVC transcriptomes revealed partially overlapping expression profiles of various ion channels and gap junction proteins which reflects their diversified functions. Lastly, we observed dynamic expression of epithelial-to-mesenchymal transition markers, as well as components of TGF-{beta}, Notch, and Wnt signaling pathways, which have been implicated in the formation of AVC conduction and cardiac valves. Our results uncovered the molecular hallmarks of the developing AVC region and demonstrated its role in the structural and electrophysiological separation between the atrium and ventricle. Author summaryThe atrioventricular canal is a structure in the embryonic heart which separates the atrium from the ventricle. It gives rise to the AV node and cardiac valves - two important structures which ensure unidirectional blood flow between heart chambers. The AV node serves to regulate the propagation of electrical impulses between the two chambers, such that they contract consecutively. Using the zebrafish as model organism, we performed gene expression profiling and characterized electrical conduction patterns between the sinoatrial primary pacemaker and AVC. We discovered that the zebrafish AVC possesses similar features to the mammalian AV node, including slow conduction, inherent pacemaking activity, and the expression of conserved developmental genes. The molecular profile of the AVC is distinct from that of the sinoatrial pacemaker, which reflects their distinct roles. In addition, we found that genes regulating cardiac valve development were also expressed in the AVC, illustrating the importance of this region for establishing both electrophysiological and structural separation between the heart chambers. Besides establishing conserved aspects between zebrafish and mammalian conduction system, the data generated in this study constitutes a valuable resource for studying AVC development and discovery of novel candidate genes implicated in regulating cardiac rhythm and cardiac valve formation.

developmental biology↗

bioRxiv2021

Piezo1 and BKCa channels in human atrial fibroblasts: interplay and remodelling in atrial fibrillation

AimsAtrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging population of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC-presence and -activity associated with AF. Methods and ResultsUsing primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC-signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline and iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent big potassium channels, BKCa. In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. ConclusionsHuman atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical interactions between the two channel proteins.

molecular biology↗