Biology subjects

Tibbits, G. F.

Publications and source records attributed to Tibbits, G. F..

11 recordsLinked to original sources

bioRxiv2026

Programmed electrical stimulation in human iPSC-derived cardiomyocytes reveals mechanisms of lethal arrhythmias in Calcium Release Deficiency Syndrome

BackgroundCalcium release deficiency syndrome (CRDS) is a recently described inherited channelopathy caused by loss-of-function variants in RYR2. Clinically, CRDS patients present with lethal ventricular arrhythmias which are not reproduced on exercise stress testing, unlike catecholaminergic polymorphic ventricular tachycardia. A hallmark trigger identified for CRDS mimics a long-burst, long-pause, short-coupled extra-stimulus (LBLPS) programmed electrical stimulation protocol, which was experimentally validated in humans and mouse models. Moreover, application of a long-burst, long-pause (LBLP) protocol alone can induce an abnormal repolarization on the first sinus beat that is unique to CRDS. However, the electrophysiological basis of CRDS in human cardiac tissue, including other triggers, are not fully understood, and whether clinically relevant arrhythmias can be observed in human stem cell models remains unknown. MethodsWe performed electrophysiological and arrhythmia inducibility studies using clinically relevant programmed electrical stimulation protocols in two-dimensional cardiac tissue generated from metabolically matured human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRDS variant RyR2-E4146D. High spatiotemporal optical mapping and multielectrode arrays were used for electrophysiological phenotyping. ResultsAt baseline, E4146D+/- monolayers showed no arrhythmias, similar to controls. During rapid pacing, E4146D+/- promoted electrical vulnerability by reducing the threshold for action potential duration (APD) alternans and Ca2+ alternans and increasing the propensity for spatial discordance of alternans. In response to LBLP pacing, E4146D+/- monolayers often demonstrated an abnormal repolarization response characterized by spatially dispersed APD prolongation and large Ca2+ release. Notably, LBLPS pacing produced early-after depolarization (EAD)-driven triggered activity resulting in re-entrant tissue conduction patterns, explaining the short-coupled ectopy driven arrhythmias seen in CRDS patients. Similar arrhythmias were observed when EADs developed during spatially discordant alternans. Lastly, flecainide showed efficacy in suppressing arrhythmia inducibility for the here studied variant. ConclusionsWe developed the first hiPSC model for CRDS which recapitulates clinically observed and inducible arrhythmias. Our model provides novel insights into tissue-level, re-entrant arrhythmias, which are initiated by EADs during electrically vulnerable states in CRDS human cardiac tissue and can be suppressed by flecainide. This model provides the framework for studying other CRDS variants and complex arrhythmias in hiPSC-CMs and establishes a human-based new approach method (NAM) for drug and gene therapy development for CRDS. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABS{blacksquare} We developed the first human stem cell-derived cardiomyocyte (hiPSC-CM) tissue model for calcium release deficiency syndrome (CRDS) which recapitulates its hallmark clinical features, including inducible ventricular arrhythmias with programmed electrical stimulation and post-pacing repolarization abnormalities. {blacksquare}Using genome edited and metabolically matured hiPSC-CMs combined with high spatiotemporal optical mapping, we show that tissue-level arrhythmias are initiated by early-after depolarizations (EADs) which develop during electrically vulnerable states, leading to re-entrant conduction patterns. We comprehensively characterize the features of EAD-induced triggered activity, showing that these ectopic beats promote re-entry through slower conduction velocities and shorter action potential durations. This uncovers how EAD-induced short-coupled ectopy leads to malignant ventricular arrhythmias in CRDS patients, and establishes the phenotype for future hiPSC-CM investigations. {blacksquare}We identified flecainide as an effective agent in suppressing arrhythmias on single cell and tissue levels in hiPSC-CMs for this CRDS variant, reproducing clinical results. What are the clinical implications?{blacksquare} CRDS has only recently been described as a unique channelopathy caused by loss-of-function RYR2 variants, and much of its triggers and mechanisms in human cardiomyocytes remain unclear. The arrhythmias observed are often not related to exercise, and exercise stress testing does not reproduce these abnormalities. No human models exist to date which closely recapitulate the triggers shown to induce tissue-level arrhythmias in patients and mouse models. Our model demonstrates that programmed electrical stimulation, without pharmacological {beta}-adrenergic stimulation, can reliably induce the same arrhythmias seen clinically, enabling accurate disease modeling and drug development. {blacksquare}Combining programmed electrical stimulation in cardiac tissue derived from genome-edited hiPSC-CMs with high spatiotemporal optical mapping is a robust and novel approach to identify the mechanisms of complex, tissue-level arrhythmias which remain underexplored, such as short-coupled ventricular fibrillation, in a patient-specific and translational manner.

cell biology↗

bioRxiv2025

Sex- and ketogenesis-dependent effects of intermittent fasting against diet-induced obesity and fatty liver disease

ABSTRACTSIntermittent fasting (IF) improves metabolic health, yet the requirement for hepatic ketogenesis in mediating these benefits remains unclear. Here, we investigated how hepatic ketogenesis contributes to the metabolic and hepatic effects of IF in male and female mice. In the human liver, ketogenesis-associated genes showed sex-dependent correlations with inflammatory and fibrotic pathways. In mice, fasting increased circulating ketone bodies, with females exhibiting a greater rise, indicating intrinsic sex differences in ketone metabolism. IF reduced body weight and adiposity in both sexes, and these systemic benefits persisted despite antisense oligonucleotide (ASO)-mediated knockdown of hepatic Hmgcs2. In contrast, hepatic benefits were sex- and ketogenesis-dependent. IF markedly reduced steatosis and fibrosis in male mice, but these improvements were attenuated or abolished when hepatic ketogenesis was disrupted. Female mice showed minimal hepatic benefit from IF and displayed heightened susceptibility to steatosis, fibrosis, and inflammatory activation under ketogenic insufficiency. Single-cell transcriptomic analyses identified neutrophils and myofibroblasts as key responders to hepatocyte-derived ketone bodies, and IF suppressed neutrophil-driven inflammatory signaling in a ketogenesis-dependent manner in males but not females. Together, these findings demonstrate that while systemic metabolic improvements from IF are largely ketogenesis-independent, the hepatic anti-steatotic and anti-fibrotic effects of IF are sexually dimorphic and require intact hepatic ketogenesis.

physiology↗

bioRxiv2025

Conservation and divergence of transcriptional heterogeneity in the cardiac conduction system

The cardiac conduction system (CCS) consists of specialized cardiomyocytes that initiate and propagate electrical activity through the heart. While the transcriptional programs underlying CCS development and function have been studied within individual species, how these programs compare across species and developmental stages remains unclear. Here, we present a comprehensive cross-species and cross-stage analysis of the CCS transcriptome using single-cell/single-nucleus RNA sequencing and spatial transcriptomic datasets from human, mouse, rat, zebrafish, and medaka hearts. We identify shared and species-or stage-specific gene expression patterns, spanning CCS-wide, zonal-, and component-level features, as well as conserved gene regulatory networks across the species. Many conserved genes are associated with human CCS function and related disorders, highlighting their translational relevance for conduction disease. This work refines the molecular characterization of CCS cell types across vertebrates and provides a resource for advancing our understanding of CCS development, function, and pathology.

genomics↗

bioRxiv2025

Molecular rewiring of human induced pluripotent stem cell-derived cardiomyocytes during metabolic maturation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used to model cardiac development and inherited cardiomyopathies, yet their immature metabolic state limits interpretation of disease-associated molecular programs. While multiple strategies promote structural and functional maturation, less is known about the molecular regulation of metabolic maturation as a distinct developmental transition. Here, we examine how metabolic maturation reshapes molecular and metabolic states in wild-type and TNNT2-linked hypertrophic cardiomyopathy (HCM) hiPSC-CMs. Using integrated transcriptomic, chromatin accessibility, proteomic, and metabolic profiling, we define the molecular trajectory associated with metabolic maturation in wild-type hiPSC-CMs, characterized by coordinated transcriptional and epigenetic remodeling, enhanced mitochondrial oxidative metabolism, and progressive suppression of mTORC1 signaling. In contrast, hiPSC-CMs carrying TNNT2 HCM variants (I79N+/- and R278C+/-) exhibit variant-specific deviations from this metabolic maturation trajectory. While early CM differentiation is largely preserved, metabolic maturation reveals defects in mitochondrial respiration and chromatin organization, with the more clinically severe I79N+/- variant showing sustained metabolic impairment. We further find that mTORC1 activity is temporally misregulated during metabolic maturation in HCM hiPSC-CMs, with reduced signaling at early stages and normalization at later time points. Pharmacological inhibition of mTORC1 with rapamycin partially improves disease-associated protein expression signatures in the I79N+/- variant, particularly when applied during early differentiation. Together, these findings demonstrate that TNNT2-linked HCM involves disrupted metabolic maturation programs coupled to altered gene regulatory states, highlighting metabolic maturation as a critical context for studying cardiomyopathy-associated molecular phenotypes in hiPSC-CMs.

molecular biology↗

bioRxiv2024

Developing a Soft Micropatterned Substrate to Enhance Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSC-CM immaturity by introducing a novel physiologically relevant micropatterned substrate for long-term culture and maturation. A novel microfabrication technique combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue, respectively. Platinum electrodes integrated into the cell culture chamber enabled pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca2+ transient, sarcomere orientation, distribution, and nucleus aspect ratio are analyzed in a 2D hiPSC-CM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSC-CMs for three weeks on a micropatterned PDMS substrate (2.5-5 {micro}m deep, 20 {micro}m center-to-center spacing of grooves, 2-5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, nucleus aspect ratio, contractility, and cytosolic Ca2+ transient. The study provides significant insights into substrate stiffness effects on hiPSC-CM contractility and Ca2+ transient at immature and mature states. Maximum contractility and fastest Ca2+ transient kinetics occur in mature hiPSC-CMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. This new substrate offers a promising platform for disease modeling and therapeutic interventions.

bioengineering↗

bioRxiv2023

Molecular and metabolomic characterization of hiPSC-derived cardiac fibroblasts transitioning to myofibroblasts

1.Mechanical stress and pathological signaling trigger the activation of fibroblasts to myofibroblasts, which impacts extracellular matrixcomposition, disrupts normal wound healing,andcan generate deleterious fibrosis (Bohl et al., 2008; Sutton and Sharpe, 2000). Myocardial fibrosis independently promotes cardiac arrhythmias, sudden cardiac arrest, and contributes to the severity of heart failure (Frangogiannis, 2021). Fibrosis can also alter cell-to-cell communication and increase myocardial stiffness which eventually may lead to lusitropic and inotropic cardiac dysfunction (PMID: 33135058). Human induced pluripotent stem cell derived cardiac fibroblasts (hiPSC-CFs) have the potential to enhance clinical relevance in precision disease modeling by facilitating the study of patient-specific phenotypes. However, it is unclear whether hiPSC-CFs can be activated to become myofibroblasts akin to primary cells, and the key signaling mechanisms in this process remain unidentified. We hypothesize that the passaging of hiPSC-CFs, like primary cardiac fibroblasts, induces specific genes required for myofibroblast activation and increased mitochondrial metabolism. Passaging of hiPSC-CFs from passage 0 to 3 (P0 to P3) and treatment of P0 with TGF{beta}1 was associated with a gradual induction of genes to initiate the activation of these cells to myofibroblasts, including collagen, periostin, fibronectin, and collagen fiber processing enzymes with concomitant downregulation of cellular proliferation markers. Most importantly, canonical TGF{beta}1 and Hippo signaling component genes including TAZ were influenced by passaging hiPSC-CFs. Seahorse assay revealed that passaging and TGF{beta}1 treatment increased mitochondrial respiration, consistent with fibroblast activation requiring increased energy production, whereas treatment with the glutaminolysis inhibitor BPTES completely attenuated this process. Based on these data, the hiPSC-CF passaging enhanced fibroblast activation, activated fibrotic signaling pathways, and enhanced mitochondrial metabolism approximating what has been reported in primary cardiac fibroblasts. Thus, hiPSC-CFs may provide an accurate in vitro preclinical model for the cardiac fibrotic condition, which may facilitate the identification of putative anti-fibrotic therapies, including patient-specific approaches. HighlightsO_LIPassaging promotes the activation of fibroblasts to myofibroblasts. C_LIO_LITGF{beta}1 treatment activates the fibroblasts, but their expression profile was uniquely different from myofibroblasts. C_LIO_LIHigh energy requiring fibroblast activation is dependent on glutaminase-based mitochondrial metabolism. C_LIO_LIPassaging induces TGF{beta}1 and Hippo signaling pathways in activated fibroblasts and myofibroblasts. C_LI Graphical Abstract CaptionProbing the activation of fibroblasts to myofibroblasts is key in ECM remodeling processes to avoid fibrosis-related adverse complications, and to better understand disease pathology. Here we report that passaging of hiPSC-derived cardiac fibroblasts promotes fibroblast activation along with a gradual shift in gene expression and metabolic changes towards myofibroblasts. TGF{beta}1 treatment activates non-passaged fibroblasts, but they are dissimilar to myofibroblasts. The energy-intensive fibroblast to myofibroblast activation process is dependent on glutaminase-mediated mitochondrial metabolism and is prevented by treatment with GLS-1 inhibitor BPTES. Our work demonstrates that hiPSC-CFs can offer a preclinical model analogous to primary cardiac fibroblasts that is comparable with passage-mediated myofibroblast activation and increased mitochondrial metabolism. hiPSC-CFs may also facilitate patient-specific novel anti-fibrosis drug screening and disease management. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/561455v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@139f3a9org.highwire.dtl.DTLVardef@1abea35org.highwire.dtl.DTLVardef@19d7bfborg.highwire.dtl.DTLVardef@369e9e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Mechanisms of Pathogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant R278C+/- During Development

Hypertrophic cardiomyopathy (HCM) is one of the most common heritable cardiovascular diseases and variants of TNNT2 (cardiac troponin T) are linked to increased risk of sudden cardiac arrest despite causing limited hypertrophy. In this study, a TNNT2 variant, R278C+/-, was generated in both human cardiac recombinant/reconstituted thin filaments (hcRTF) and human-induced pluripotent stem cells (hiPSCs) to investigate the mechanisms by which the R278C+/- variant affects cardiomyocytes at the proteomic and functional levels. The results of proteomics analysis showed a significant upregulation of markers of cardiac hypertrophy and remodeling in R278C+/- vs. the isogenic control. Functional measurements showed that R278C+/- variant enhances the myofilament sensitivity to Ca2+, increases the kinetics of contraction, and causes arrhythmia at frequencies >75 bpm. This study uniquely shows the profound impact of the TNNT2 R278C+/- variant on the cardiomyocyte proteomic profile, cardiac electrical and contractile function in the early stages of cardiac development. Translational PerspectiveHypertrophic cardiomyopathy (HCM) is the leading known cause of sudden cardiac arrest in the young. Thin-variant associated HCM variants make up to 15% of familial HCM yet their molecular mechanisms remain less clear relative to thick filament variants. Here, we employ computational modeling, human cardiac recombinant/reconstituted thin filaments (hcRTF), and hiPSC-CMs to study the thin filament TNNT2 R278C+/- variant, revealing its extensive pathogenicity and potential mechanisms by which it can lead to HCM and sudden death. Mavacamten, the recently FDA-approved treatment, was effective in alleviating contractile dysfunction in TNNT2 R278C+/- hiPSC-CMs, positing it as a potential therapy for thin filament HCM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/542948v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@102a8b0org.highwire.dtl.DTLVardef@190e356org.highwire.dtl.DTLVardef@13d3f9aorg.highwire.dtl.DTLVardef@1a9c33_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Patient and cell-type specific hiPSC-modeling of a truncating titin variant associated with atrial fibrillation

BackgroundProtein truncating mutations in the titin gene are associated with increased risk of atrial fibrillation (AF). However, little is known regarding the underlying pathophysiology. MethodsWe identified a heterozygous titin truncating variant in a patient with unexplained early-onset AF using whole exome sequencing. We used atrial and ventricular patient induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), CRISPR/Cas9 genetic correction, and engineered heart tissue (EHT) constructs to evaluate the impact of the titin truncating variant on electrophysiology, sarcomere structure, contractility, and gene expression. ResultsWe generated atrial and ventricular iPSC-CMs from the AF patient with the titin truncating variant and a CRISPR/Cas9 genome corrected isogenic control. We demonstrate that the titin truncating variant increases susceptibility to pacing-induced arrhythmia (prevalence of arrhythmogenic phenotypes, 85.7% versus 14.2%; P = 0.03), promotes sarcomere disorganization (mean {+/-} SEM, 66.3 {+/-} 6.8% versus 88.0 {+/-} 2.9%; P = 0.04) in atrial iPSC-CMs, and reduces contractile force (0.013 {+/-} 0.003 mN versus 0.027 {+/-} 0.004 mN; P < 0.01) in atrial EHTs compared to isogenic controls. In ventricular iPSC-CMs, this variant led to altered electrophysiology (90.0% versus 33.3%; P = 0.02) and sarcomere organization (62.0 {+/-} 3.9% versus 82.9 {+/-} 2.9%; P < 0.01) with no change in EHT contractility compared to isogenic controls. RNA-sequencing revealed an upregulation of cell adhesion and extracellular matrix genes in the presence of the titin truncating variant for both atrial and ventricular EHTs. ConclusionsIn a patient with early-onset unexplained AF and normal ventricular function, iPSC-CMs with a titin truncating variant showed structural and electrophysiological abnormalities in both atrial and ventricular preparations, while only atrial EHTs demonstrated reduced contractility. Whole transcriptome sequencing showed upregulation of genes involved in cell-cell and cell-matrix interactions in both atrial and ventricular EHTs. Together, these findings suggest titin truncating variants promote the development of AF through remodeling of atrial cardiac tissue and provide insight into the chamber-specific effects of titin truncating variants.

cell biology↗

bioRxiv2021

The effect of magnesium on calcium binding to cardiac troponin C related hypertrophic cardiomyopathy mutants

Cardiac troponin C (cTnC) is the calcium (Ca2+) sensing component of the troponin complex. Binding of Ca2+ to cTnC triggers a cascade of myofilament conformational changes that culminate in force production. Mutations in cTnC linked to hypertrophic myocardial myopathy (HCM) induce a a greater degree and duration of Ca2+ binding, which may underly the hypertrophic phenotype. Recent evidence from our laboratories demonstrated novel modifications of cTnC Ca2+ binding by cellular magnesium (Mg2+) that we hypothesize may be of significance in promoting HCM. Regulation of contraction has long been thought to occur exclusively through Ca2+ binding to site II of cTnC. However, abundant cellular Mg2+ is a potential competitor for binding to the same sites; work by several groups also suggests this is possible. We have used isothermal titration calorimetry (ITC) to explore the thermodynamic properties associated with the interaction between Ca2+/Mg2+ and site II of cTnC; these experiments demonstrated that physiological concentrations of Mg2+ may compete with Ca2+ to bind site II of cTnC. In experiments reported here, we studied a series of mutations in cTnC thought to be causal in HCM. Three mutants (A8V, L29Q, and A31S) slightly elevated the affinity for both Ca2+ and Mg2+, whereas other mutants (L48Q, Q50R, and C84Y), that are closer to the C-terminal domain and surrounding the EF hand binding motif of site II had a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg2+ in modifying the Ca2+ affinity ofcTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg2+ at baseline conditions and when elevated on the control of the dynamics of contraction by modifications in the Ca2+ binding properties of cTnC.

cell biology↗

bioRxiv2020

Drug Screening Platform Using Human Induced Pluripotent Stem Cell-Derived Atrial Cardiomyocytes and Optical Mapping

AO_SCPLOWBSTRACTC_SCPLOWCurrent drug development efforts for the treatment of atrial fibrillation (AF) are hampered by the fact that many preclinical models have been unsuccessful in reproducing human cardiac atrial physiology and its response to medications. In this study, we demonstrated an approach using human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes (hiPSC-aCMs and hiPSC-vCMs, respectively) coupled with a sophisticated optical mapping system for drug screening of atrial-selective compounds in vitro. We optimized differentiation of hiPSC-aCMs by modulating the WNT and retinoid signalling pathways. Characterization of the transcriptome and proteome revealed that retinoic acid pushes the differentiation process into the atrial lineage and generated hiPSC-aCMs. Functional characterization using optical mapping showed that hiPSC-aCMs have shorter action potential durations and faster Ca2+ handling dynamics compared to hiPSC-vCMs. Furthermore, pharmacological investigation of hiPSC-aCMs captured atrial-selective effects by displaying greater sensitivity to atrial-selective compounds 4-aminopyridine, AVE0118, UCL1684, and vernakalant when compared to hiPSC-vCMs. These results established that a model system incorporating hiPSC-aCMs combined with optical mapping is well-suited for pre-clinical drug screening of novel and targeted atrial selective compounds.

pharmacology and toxicology↗

bioRxiv2020

Binding of Calcium and Magnesium to Cardiac Troponin C

Cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) which bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. The latter binding initiates a series of conformational changes that culminate in force production. We have quantified the interaction between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and Thermodynamic Integration simulations. Direct and competitive binding titrations using wild type and a double mutant that significantly reduces binding to site II demonstrated that physiologically relevant concentrations of both Ca2+/Mg2+ interact with the same locus. Cytosolic free Mg2+ (~1 mM) could occupy a significant population of available site II, as this concentration of Mg2+ decreased the affinity for Ca2+ 1.4-fold. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart at baseline and in diseased states which decrease or increase availability of Mg2+ such as secondary hyperparathyroidism or ischemia, respectively.

cell biology↗