Biology subjects

Louch, W. E.

Publications and source records attributed to Louch, W. E..

9 recordsLinked to original sources

bioRxiv2025

Reduced gap junction coupling amplifies the effects of cardiomyocyte variability and destabilizes the heartbeat

Cardiomyocytes exhibit significant cell-to-cell variability due to differences in protein expression and post-tra nslational modifications in both the cell membrane and the intracellular machinery. Resulting variability in action potential propagation and configuration have been proposed to promote arrhythmia. However, such effects may be suppressed by tight electrical coupling of cells in the healthy heart, but not during pathological conditions where gap junction function is impaired. To investigate this question, we employed a cell-based mathematical model of cardiac electrophysiology, in which we systematically modified both the properties of individual cells within the array, and inter-cellular electrical connectivity (gap junctions). Despite the inclusion of marked variation in properties between cells, we observed electrical homogeneity across the array when cells were well coupled. In contrast, lower and/or more variable gap junction connectivity resulted in nonhomogeneous action potential configuration, and irregular timing of both the depolarizing and repolarizing electrical wavefronts. Pro-arrhythmic early after-depolarizations also occurred under these conditions, linked to reopening of L-type calcium channels. These effects were effectively dampened in highly coupled cells. Nevertheless, baseline differences in calcium homeostasis were not negated by gap junction coupling, indicating a limit to which electrical connections can ho-mogenize mechanical function. There are also physical limits to electrical convergence, as we observed that action potential differences persisted at the edges and corners of the array where there are fewer electrical contacts with neighbouring cells. This finding may have implications for arrhythmic susceptibility in the border zone neighbouring an infarction. In summary, our findings underscore the critical role of intercellular coupling in maintaining cardiac stability and highlight the importance of studying cardiomyocytes within a syncytium rather than in isolation.

biophysics↗

bioRxiv2024

Enhanced Ca2+-Driven Arrhythmias in Female Patients with Atrial Fibrillation: Insights from Computational Modeling

Background and AimsSubstantial sex-based differences have been reported in atrial fibrillation (AF), with female patients experiencing worse symptoms, increased complications from drug side effects or ablation, and elevated risk of AF-related stroke and mortality. Recent studies revealed sex-specific alterations in AF-associated Ca2+ dysregulation, whereby female cardiomyocytes more frequently exhibit potentially proarrhythmic Ca2+-driven instabilities compared to male cardiomyocytes. In this study, we aim to gain a mechanistic understanding of the Ca2+-handling disturbances and Ca2+-driven arrhythmogenic events in males vs females and establish their responses to Ca2+-targeted interventions. Methods and ResultsWe incorporated known sex differences and AF-associated changes in the expression and phosphorylation of key Ca2+-handling proteins and in ultrastructural properties and dimensions of atrial cardiomyocytes into our recently developed 3D atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca2+-handling processes. Our simulations of quiescent cardiomyocytes show increased incidence of Ca2+ sparks in female vs male myocytes in AF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to develop pacing-induced spontaneous Ca2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca2+ transients compared with the male model. Parameter sensitivity analysis uncovered precise arrhythmogenic contributions of each component that was implicated in sex and/or AF alterations. Specifically, increased ryanodine receptor phosphorylation in female AF cardiomyocytes emerged as the major SCR contributor, while reduced L-type Ca2+ current was protective against SCRs for male AF cardiomyocytes. Furthermore, simulations of tentative Ca2+-targeted interventions identified potential strategies to attenuate Ca2+-driven arrhythmogenic events in female atria (e.g., t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca{superscript 2}-ATPase), and revealed enhanced efficacy when applied in combination. ConclusionsOur sex-specific computational models of human atrial cardiomyocytes uncover increased propensity to Ca2+-driven arrhythmogenic events in female compared to male atrial cardiomyocytes in AF, and point to combined Ca2+-targeted interventions as promising approaches to treat AF in female patients. Our study establishes that AF treatment may benefit from sex-dependent strategies informed by sex-specific mechanisms. Translational perspectiveAccumulating evidence demonstrates substantial sex-related differences in atrial fibrillation (AF), which is the most common arrhythmia, with female patients faring worse with the condition. By integrating known sex-differential components into our computational atrial cardiomyocyte model we found that female atrial cardiomyocytes in AF exhibit greater propensity to develop Ca2+-driven arrhythmia than male cardiomyocytes. Model analyses provided novel mechanistic insights and suggested strategies such as t-tubule restoration, correction of Ca2+-handling disturbances, and the combination of both, as promising approaches to treat AF in female patients. Our study uncovers and validate sex-specific AF mechanisms and inform the development of targeted anti-AF strategies. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/583217v2_figa1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@d3ec3borg.highwire.dtl.DTLVardef@13a2c7borg.highwire.dtl.DTLVardef@3e4605org.highwire.dtl.DTLVardef@6726b4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Sex-specific 3D spatiotemporal models of human atrial cardiomyocyte Ca2+ signaling reveal a greater propensity to develop Ca2+-driven arrhythmic events in female vs male atrial cardiomyocytes in AF. Model analysis links sex-specific AF remodeling to arrhythmogenic mechanisms. AF, atrial fibrillation; SCR, spontaneous Ca2+ release; CaT, cytosolic Ca2+ transient; RyR2-P, phosphorylated ryanodine receptor type 2 (RyR2); CSQ, calsequestrin; LTCC, L-type Ca2+ channel; PLB, phospholamban; SERCA, sarcoendoplasmic reticulum Ca2+ ATPase; SR, sarcoplasmic reticulum. C_FIG

physiology↗

bioRxiv2023

Analysis of RyR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D MINFLUX microscopy

The cardiac type 2 ryanodine receptor (RyR2) is a large homotetramer of a [~]560 kD subunit and is the molecular pathway through which the majority of Ca2+ enters the cytosol during cardiac activation. It constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative giving rise to local release events termed Ca2+ sparks. Accordingly, the molecular distribution of RyR2 in cardiac myocytes has been of great interest. Here we present the first purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX fluorescence super-resolution microscopy. We demonstrate that by using single-domain antibodies (sdABs) against fluorescent protein domains in engineered RyR2 fluorescent protein fusions we can determine the location of individual RyR2 subunits with high precision ([~]3 nm) in all directions. Combining MINFLUX with DNA-PAINT, to maximize detection efficiency, we measured in situ labeling efficiencies using NPC structures as reference and regularly achieved efficiencies around 50%, which would translate to RyR2 detection efficiencies close to 95%, i.e. the probability that at least one subunit is detected, if target accessibility is similar. Using this approach, we detect dense and extended RyR2 expression in HEK cells with some clusters spanning several micrometres in extent and containing several hundred RyR2s. Similarly, myocytes from PA-RFP RyR2 mice contained large clusters containing many tens of RyR2s. The new data also suggests a resolution to apparent discrepancies between previous data from electron microscopy and super-resolution data. The methodology developed here will be critical to reveal the full complexity of RyR2 and related Ca2+ handling proteins in 3D as well as their relationship to contractile function. Our new approaches should be applicable to other multi-subunit complexes in cardiac muscle and other cell types.

physiology↗

bioRxiv2022

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: I. Transverse-axial tubule variation

Intracellular calcium (Ca2+) cycling is tightly regulated in the healthy heart ensuring effective contraction. This is achieved by transverse (t)-tubule membrane invaginations that facilitate close coupling of key Ca2+-handling proteins such as the L-type Ca2+ channel (LCC) and Na+-Ca2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca2+ store. Though less abundant and regular than in the ventricle, t-tubules also exist in atrial myocytes as a network of transverse invaginations with axial extensions known as the transverse-axial tubule system (TATS). In heart failure and atrial fibrillation there is TATS remodeling that is associated with aberrant Ca2+-handling and Ca2+-induced arrhythmic activity, however the mechanism underlying this is not fully understood. To address this, we developed a novel 3D human atrial myocyte model that couples electrophysiology and Ca2+-handling with variable TATS organization and density. We extensively parameterized and validated our model against experimental data to build a robust tool examining TATS regulation of subcellular Ca2+ release. We found that varying TATS density and thus the localization of key Ca2+-handling proteins has profound effects on Ca2+ handling. Following TATS loss there is reduced NCX that results in increased cleft Ca2+ concentration through decreased Ca2+ extrusion. This elevated Ca2+ increases RyR open probability causing spontaneous Ca2+ releases and promotion of arrhythmogenic waves (especially in the cell interior) that leads to voltage instabilities through delayed afterdepolarizations. In summary, this study demonstrates a mechanistic link between TATS remodeling and Ca2+-driven proarrhythmic behavior that likely reflects the arrhythmogenic state observed in disease. Key PointsO_LITransverse-axial tubule systems (TATS) modulate Ca2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodeling in heart failure and atrial fibrillation associated with altered Ca2+ cycling and subsequent arrhythmogenesis. C_LIO_LITo investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca2+ handling governed by the TATS. C_LIO_LISimulated TATS loss causes diastolic Ca2+ and voltage instabilities through reduced NCX-mediated Ca2+ removal, cleft Ca2+ accumulation and increased RyR open probability, resulting in spontaneous Ca2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. C_LIO_LIAt fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca2+ releases are larger and more frequent in the cell interior than at the periphery. C_LIO_LIOur work provides mechanistic insight into how atrial TATS remodeling can lead to Ca2+- driven instabilities that may ultimately contribute to the arrhythmogenic state in disease. C_LI

physiology↗

bioRxiv2022

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: II. Ca2+-handling protein variation

Disruption of the transverse-axial tubule system (TATS) in diseases such as heart failure and atrial fibrillation occurs in combination with changes in the expression and distribution of key Ca2+- handling proteins. Together this ultrastructural and ionic remodeling is associated with aberrant Ca2+ cycling and electrophysiological instabilities that underly arrhythmic activity. However, due to the concurrent changes in TATs and Ca2+-handling protein expression and localization that occur in disease it is difficult to distinguish their individual contributions to the arrhythmogenic state. To investigate this, we applied our novel 3D human atrial myocyte model with spatially detailed Ca2+ diffusion and TATS to investigate the isolated and interactive effects of changes in expression and localization of key Ca2+-handling proteins and variable TATS density on Ca2+- handling abnormality driven membrane instabilities. We show that modulating the expression and distribution of the sodium-calcium exchanger, ryanodine receptors, and the sarcoplasmic reticulum (SR) Ca2+ buffer calsequestrin have varying pro and anti-arrhythmic effects depending on the balance of opposing influences on SR Ca2+ leak-load and Ca2+-voltage relationships. Interestingly, the impact of protein remodeling on Ca2+-driven proarrhythmic behavior varied dramatically depending on TATS density, with intermediately tubulated cells being more severely affected compared to detubulated and densely tubulated myocytes. This work provides novel mechanistic insight into the distinct and interactive consequences of TATS and Ca2+-handling protein remodeling that underlies dysfunctional Ca2+ cycling and electrophysiological instability in disease. Key PointsO_LIIn our companion paper we developed a 3D human atrial myocyte model, coupling electrophysiology and Ca2+ handling with subcellular spatial details governed by the transverse-axial tubule system (TATS). C_LIO_LIHere we utilize this model to mechanistically examine the impact of TATS loss and changes in the expression and distribution of key Ca2+-handling proteins known to be remodeled in disease on Ca2+ homeostasis and electrophysiological stability. C_LIO_LIWe demonstrate that varying the expression and localization of these proteins has variable pro- and anti-arrhythmic effects with outcomes displaying dependence on TATS density. C_LIO_LIWhereas detubulated myocytes typically appear unaffected and densely tubulated cells seem protected, the arrhythmogenic effects of Ca2+ handling protein remodeling are profound in intermediately tubulated cells. C_LIO_LIOur work shows the interaction between TATS and Ca2+-handling protein remodeling that underlies the Ca2+-driven proarrhythmic behavior observed in AF and may help to predict the effects of antiarrhythmic strategies at varying stages of ultrastructural remodeling. C_LI

physiology↗

bioRxiv2022

Prolonged β-Adrenergic Stimulation Disperses Ryanodine Receptor Clusters in Cardiomyocytes: Implications for Heart Failure

Ryanodine Receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that "dispersion" of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged {beta}-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion, and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hour triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of CaMKII reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of {beta}-adrenergic stimulation over the longer term, and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology. Significance statementThe heartbeat is triggered by the release of Ca2+ from Ryanodine Receptors (RyRs) within cardiomyocytes. Recent data indicate RyR arrangement is highly malleable. However, mechanisms controlling RyR reorganisation and the subsequent impact on Ca2+ homeostasis remain unclear. Here, we show that prolonged {beta}-adrenergic stimulation causes RyR clusters to disperse, drastically altering the frequency and kinetics of Ca2+ release events called "Ca2+ sparks" in a process that is dependent on CaMKII and PKA. In healthy cells, these compensatory effects protect against arrhythmogenic Ca2+ over-activity. However, during heart failure, RyR hyper-phosphorylation and dispersion impairs Ca2+ release and cardiac performance. Thus, RyR localization and function are intimately linked via channel phosphorylation which, while finely tuned in health, underlies impaired cardiac function during pathology.

cell biology↗

bioRxiv2021

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks

Super-resolution imaging techniques have provided a better understanding of the relationship between the nanoscale organization of function of ryanodine receptors (RyRs) in cardiomy-ocytes. Interestingly recent data have indicated that this relationship is disrupted in heart failure (HF), as RyRs are dispersed into smaller and more numerous clusters. However, RyRs are also hyperphosphorylated in this condition, and this is reported to occur preferentially within the cluster centre. Thus, the combined impact of RyR relocalization and sensitization on Ca2+ spark generation in failing cardiomyocytes is likely complex and these observations suggest that both the nanoscale organization of RyRs and the pattern of phosphorylated RyRs within clusters could be critical determinants of Ca2+ spark dynamics. To test this hypothesis, we used computational modeling to quantify the relationships between RyR cluster geometry, phosphorylation patterns, and sarcoplasmic reticulum (SR) Ca2+ release. We found that RyR cluster disruption results in a decrease in spark fidelity and longer sparks with a lower amplitude. Phosphorylation of some RyRs within the cluster can play a compensatory role, recovering healthy spark dynamics. Interestingly, our model predicts that such compensation is critically dependent on the phosphorylation pattern, as phosphorylation localized within the cluster center resulted in longer Ca2+ sparks and higher spark fidelity compared to a uniformly distributed phosphorylation pattern. Our results strongly suggest that both the phosphorylation pattern and nanoscale RyR reorganization are critical determinants of Ca2+ dynamics in HF. Significance StatementRyRs are ion channels located on the membrane of the sarcoplasmic reticulum that are responsible for an increase in cytosolic Ca2+ during cell excitation. Here, we investigate how the geometry of RyR clusters combined with spatial phosphorylation patterns impacts on Ca2+ spark generation and kinetics. The findings from our study show that both phosphorylation pattern and RyR cluster shape and dispersion have implications on Ca2+ spark activity and provide insights into altered Ca2+ dynamics during HF.

biophysics↗

bioRxiv2018

Matched-filter based algorithm for sub- to multi-cellular classification of myocyte tubule-system

In mammalian ventricular cardiomyocytes, invaginations of the surface membrane form the transverse tubular system (T-system) which consists of transverse tubules (TTs) that align with sarcomeres and Z-lines as well as longitudinal tubules (LTs) that are present between Z-lines in some species. In many cardiac disease etiologies the T-system is perturbed, which is believed to promote spatially heterogeneous, dyssynchronous Ca2+ release and inefficient contraction. In general, T-system characterization approaches have been directed primarily at isolated cells and do not detect subcellular T-system heterogeneity. Here we present MatchedMyo, a matched-filter based algorithm for subcellular T-system characterization in isolated cardiomyocytes and millimeter-scale myocardial sections. The algorithm utilizes "filters" representative of TTs, LTs, and T-system absence. Application of the algorithm to cardiomyocytes isolated from rat disease models of myocardial infarction (MI), dilated cardiomyopathy induced via aortic banding (AB), and sham surgery confirmed and quantified heterogeneous T-system structure and remodeling. Cardiomyocytes from post-MI hearts exhibited increasing T-system disarray as proximity to the infarct increased. We found significant (p<0.05, Welchs t-test) increases in LT density within cardiomyocytes proximal to the infarct (12{+/-}3%, data reported as mean {+/-} SD, n=3) vs. sham (4{+/-}2%, n=5), but not distal to the infarct (7{+/-}1%, n=3). The algorithm also detected decreases in TTs within 5{degrees} of the myocyte minor axis for isolated AB (36{+/-}9%, n=3) and MI cardiomyocytes located intermediate (37{+/-}4%, n=3) and proximal (34{+/-}4%, n=3) to the infarct vs. sham (57{+/-}12%, n=5). Application of bootstrapping to rabbit MI tissue revealed distal sections comprised 18.9{+/-}1.0% TTs while proximal sections comprised 10.1{+/-}0.8% TTs (p<0.05), a 46.6% decrease. The matched filter approach therefore provides a robust and scalable technique for T-system characterization from isolated cells through millimeter-scale myocardial sections.

biophysics↗

bioRxiv2018

TRPML1-mediated Modulation of Dense-core Granules Tunes Functional Potential in NK Cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca2+ stores in primary NK cells reduced both target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

immunology↗