Mapping gene networks requires large amounts of transcriptomic data to learn the connections between genes, which impedes discoveries in settings with limited data, including rare diseases and ...diseases affecting clinically inaccessible tissues. Recently, transfer learning has revolutionized fields such as natural language understanding
and computer vision
by leveraging deep learning models pretrained on large-scale general datasets that can then be fine-tuned towards a vast array of downstream tasks with limited task-specific data. Here, we developed a context-aware, attention-based deep learning model, Geneformer, pretrained on a large-scale corpus of about 30 million single-cell transcriptomes to enable context-specific predictions in settings with limited data in network biology. During pretraining, Geneformer gained a fundamental understanding of network dynamics, encoding network hierarchy in the attention weights of the model in a completely self-supervised manner. Fine-tuning towards a diverse panel of downstream tasks relevant to chromatin and network dynamics using limited task-specific data demonstrated that Geneformer consistently boosted predictive accuracy. Applied to disease modelling with limited patient data, Geneformer identified candidate therapeutic targets for cardiomyopathy. Overall, Geneformer represents a pretrained deep learning model from which fine-tuning towards a broad range of downstream applications can be pursued to accelerate discovery of key network regulators and candidate therapeutic targets.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug ...interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (I
) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT.
We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate
expression.
Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and I
significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. I
reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application.
, essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol.
overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression.
silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin.
Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1.
Abstract
Aims
The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain ...largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation.
Methods and results
Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude.
Conclusion
This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.
Studies on animal models have shown that Irx5 is an important regulator of cardiac development and that it regulates ventricular electrical repolarization gradient in the adult heart. Mutations in ...IRX5 have also been linked in humans to cardiac conduction defects. In order to fully characterize the role of IRX5 during cardiac development and in cardiomyocyte function, we generated three genetically-modified human induced pluripotent stem cell lines: two knockout lines (heterozygous and homozygous) and a knockin HA-tagged line (homozygous).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Aims
Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, ...are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function.
Methods and results
Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression.
Conclusion
Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases.
Graphical Abstract
To the Editor: Brugada syndrome (BrS) is an inherited arrhythmic disease predisposing to sudden cardiac death (SCD), characterized by a typical electrocardiogram pattern that includes a J point ...elevation with a coved type ST segment.1 BrS is a complex genetic disease in which ∼20% of patients carry rare variants in SCN5A gene, whereas the others remain genetically unresolved.2 Despite this genetic complexity, we hypothesize that a common cellular phenotypic trait is at the root of this specific BrS ECG pattern. Furthermore, the steady-state activation and inactivation gating properties were not modified in BrS hiPSC-CMs (Figure S3A; Table S4). ...INa reduction is not a common trait of BrS hiPSC-CMs and appears to be solely associated with the presence of variants affecting SCN5A expression or function. SEE PDF The occurrence of EADs may be linked to an abnormally high density of depolarizing late sodium current (INa,L) during APs repolarizing phase.8 Accordingly, BrS hiPSC-CMs presented with a higher density of INa,L as compared to Ctrl and non-BrS hiPSC-CMs (Figure 3C,D). ...an increase in INa,L density was observed only in 6% and 12% of Ctrl and non-BrS hiPSC-CMs respectively, in accordance with their low EAD occurrence, whereas increased INa,L density was present in 50–85% of all BrS ventricular-like hiPSC-CMs, reminiscent of the high EAD occurrence (Figure 3B,E). ...the authors are grateful to the patients and families who agreed to participate in our research.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Mutations in LMNA gene, which encodes lamins A/C, cause a variety of diseases, called laminopathies. Some mutations are particularly associated with the occurrence of dilated cardiomyopathy. The ...genotype-phenotype relationship for these mutations is still unclear and there is currently no mutation-specific therapy.
Here, we focused on a mutation in the LMNA gene (c.665A>C, p.His222Pro), associated with the Emery-Dreyfuss Muscular dystrophy. We used LMNA H222P mutant induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 corrected isogenic control iPSCs to better characterize the cardiac phenotype associated with the mutation.
LMNA H222P mutant and the isogenic control iPSCs clones were differentiated into cardiomyocytes (iPSC-CMs). Ring-shaped cardiac organoids were generated to compare the contractile properties of the two clones. Calcium transients in mutant and corrected iPSC-CMs were measured by live confocal imaging. Basal and maximal mitochondrial respiration were measured by Seahorse.
iPSC-CMs were generated from the LMNA mutant and the corrected iPSCs with no difference in the differentiation yield nor in the iPSC-CMs structure. However, cardiac organoids generated with LMNA H222P iPSC-CMs showed an impaired contractility compared to control organoids. Calcium transient recordings in LMNA H222P mutant cardiomyocytes showed a significantly higher calcium transient amplitude with a significantly slower calcium re-uptake. Transcriptomic analyses suggested a global mitochondrial dysfunction and in particular an impaired mitochondrial calcium uptake with a significantly decreased expression of the mitochondrial calcium uniporter (MCU). This decrease in MCU expression was confirmed by Western blot and was accompanied by an increased MICU1: MCU ratio, as well as an increased PDH Ser232 phosphorylation, indicating a decreased mitochondrial calcium uptake in the LMNA mutant iPSC-CMs. Measurement of mitochondrial respiration using Seahorse showed lower basal and maximal respiration in LMNA H222P iPSC-CMs.
LMNA H222P mutant iPSC-CMs exhibit contractile dysfunction associated with mitochondrial dysfunction with impaired MCU complex activity, decreased mitochondrial calcium homeostasis and reduced mitochondrial energy production.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Primary aldosteronism (PA) is the most frequent form of secondary hypertension and is due to autonomous aldosterone production by the adrenal gland. During the last decades, major advances have been ...made in our understanding of the disease with the identifications of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis.
The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating calcium signaling in the cells.
We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells and activation of calcium signaling. In parallel, we have developed a mouse model expressing the a7-5HT3 receptor specifically in the adrenal cortex.
The cells expressing the a7-5HT3 receptor recapitulated the major features of KCNJ5 mutations, the most frequent genetic alteration identified in Aldosterone-Producing-Adenoma. Treatment of these cells PSEM-817 induced a significant increase in intracellular calcium concentrations, CYP11B2 mRNA expression, and aldosterone biosynthesis but did not affect neither cell proliferation nor cell death. Interestingly, gene expression analyses and steroid profiles highlighted the activation of specific signaling pathways in response to PSEM-817 and allowed us to define a sodium specific induced signature. The exploration of adult mice expressing the a7-5HT3 receptor specifically in the adrenal cortex, generated in our laboratory, is ongoing. Preliminary results showed that treatment of mice expressing the a7-5HT3 receptor with PSEM-817 for four weeks resulted in a significant increase of plasma aldosterone levels in both male and female mice.
These cell line and mouse model, in which we can modulate the intracellular calcium concentration “on demand”, are useful tools for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cardiomyocytes derived from human-induced pluripotent stem cells (iPS-CMs) have numerous advantages for drug screening and disease modeling but present with structural and functional immaturity ...features. Experiments in cardiomyocytes isolated from adult human or animal hearts are conditioned by their typical rod-shaped morphology.
Here, we aimed at improving iPS-CMs architectural maturity by using a rod-shaped cell micropatterned substrate made of repeated rectangles of 120μm-length and 30μm-width and surrounded with a chemical cell-repellent.
Cardiomyocyte differentiation was performed using a chemically defined method. Thirty-five days later the generated iPS-CMs were cultured on unpatterned vs. rod-shaped micropatterned substrates for 10 additional days. We performed molecular investigations by qPCR and immunofluorescence, and electrophysiological characterization by patch-clamp technique.
The culture of micropatterned substrates resulted in the generation of rod-shaped iPS-CMs with a 4:1 cell ratio, with a more organized troponin T staining pattern exhibiting sarcomere-type striations and higher sarcomere organization score (0.25±0.07 vs. 0.13±0.004, P<0.0001). The rod-shaped iPS-CMs also showed significant improvements in electrophysiological parameters with a decrease of the spontaneous beating rate and a more hyperpolarized membrane diastolic potential (−68.11±6.25 vs. −59.17±4.94mV, P<0.001) as compared to unpatterned iPS-CMs. Rod-shaped iPS-CMs displayed a more predominant ventricular-like type after analyzing action potential recordings (91%±1), with an increase in the percentage of late ventricular-like APs compared to unpatterned (64% vs. 29%), suggesting that rod-shaped morphology stimulates ventricular maturation. Notably, the maximum upstroke velocity (dV/dtmax) (98.09±32.63 vs. 59.71±50.43V/ms, P<0.1) and the sodium current density (INa) was increased (−89.30±25.09 vs. −64.92±21.63 pA/pF, P<0.001) in rod-shaped iPS-CMs compared to unpatterned CMs. Importantly, we found no significant change in the RNA expression of the main ion channels or in major sarcomeric components. We are now assessing the Ca2+signaling properties by confocal analysis.
We developed a novel method to generate rod-shaped iPS-CMs, leading to improved functional characteristics. Preliminary results suggest that the architectural re-arrangement is the primary driver of the improved functionality observed in micropatterned iPS-CMs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Peripartum cardiomyopathy (PPCM) is an idiopathic form of pregnancy-induced heart failure associated with preeclampsia. Circulating factors in late pregnancy are thought to contribute to both ...diseases, suggesting a common underlying pathophysiological process. However, what drives this process remains unclear. Using serum proteomics, we identified the senescence-associated secretory phenotype (SASP), a marker of cellular senescence associated with biological aging, as the most highly up-regulated pathway in young women with PPCM or preeclampsia. Placentas from women with preeclampsia displayed multiple markers of amplified senescence and tissue aging, as well as overall increased gene expression of 28 circulating proteins that contributed to SASP pathway enrichment in serum samples from patients with preeclampsia or PPCM. The most highly expressed placental SASP factor, activin A, was associated with cardiac dysfunction or heart failure severity in women with preeclampsia or PPCM. In a murine model of PPCM induced by cardiomyocyte-specific deletion of the gene encoding peroxisome proliferator-activated receptor γ coactivator-1α, inhibiting activin A signaling in the early postpartum period with a monoclonal antibody to the activin type II receptor improved heart function. In addition, attenuating placental senescence with the senolytic compound fisetin in late pregnancy improved cardiac function in these animals. These findings link senescence biology to cardiac dysfunction in pregnancy and help to elucidate the pathogenesis underlying cardiovascular diseases of pregnancy.
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