Generation of two familial hypercholesterolemia patient-specific induced pluripotent stem cell lines harboring heterozygous mutations in the LDLR gene.

Familial hypercholesterolemia (FH) is a genetic disorder affecting the metabolism of lipoprotein, characterized by elevated levels of plasma concentrations of low-density lipoprotein cholesterol (LDLC). The most common FH cause is mutations within the gene that encodes for the LDL receptor (LDLR) protein. Two induced pluripotent stem cell (iPSC) lines were generated from patients with FH, each carrying a single heterozygous mutation in the LDLR gene, one is a missense mutation, c.631C > T, and the other is a splice-site mutation, c.313 + 1G > A. Both iPSC lines exhibited strong expression of pluripotency markers, demonstrated the ability to differentiate into derivatives of the three germ layers, and maintained normal karyotypes. These derived iPSC lines represent powerful tools for in vitro modeling FH and offer a promising platform for therapeutic development.

Generation of induced pluripotent stem cell lines from patients with LQT1 caused by heterozygous mutations in the KCNQ1 gene.

Long QT Syndrome (LQTS) is a genetic heart disorder that can induce cardiac arrhythmias. The most prevalent subtype, LQT1, stems from rare variants in the KCNQ1 gene. Utilizing induced pluripotent stem cells (iPSCs) enables detailed cellular studies and personalized medicine approaches for this life-threatening condition. We generated two LQT1 iPSC lines with single nucleotide nonsense mutations, c.1031 C > T and c.1121 T > A in KCNQ1. Both lines exhibited typical iPSC morphology, expressed high levels of pluripotent markers, maintained normal karyotype, and possessed the capability to differentiate into three germ layers. These cell lines serve as important tools for investigating the biological mechanisms underlying LQT1 due to mutations in the KCNQ1 gene.

Analysis of tofacitinib safety in ulcerative colitis from the completed global clinical developmental program up to 9.2 years of drug exposure.

BACKGROUND AND AIMS: Tofacitinib is an oral Janus kinase inhibitor for the treatment of ulcerative colitis (UC). We report an integrated summary of tofacitinib safety from the completed global UC clinical program (9.2 years maximum tofacitinib exposure). METHODS: This analysis included patients receiving tofacitinib 5 or 10 mg twice daily (b.i.d.) from completed phase 2/3 placebo-controlled studies, an open-label, long-term extension study and a randomized phase 3b/4 study. Proportions and incidence rates (IRs; unique patients with events/100 patient-years [PY] of exposure) were evaluated for deaths and adverse events (AEs) of special interest (AESI). RESULTS: Overall, 1157 patients received ≥1 dose of tofacitinib 5 or 10 mg b.i.d.; 938 (81.1%) were in the predominant dose tofacitinib 10 mg b.i.d. group; 552 (47.7%) received tofacitinib for ≥2 years; total exposure: 3202.0 PY; 994 (85.9%) experienced AEs; 254 (22.0%) experienced serious AEs. Median treatment duration: 1.7 (range 0.0-9.2) years. IRs (95% CI) for combined tofacitinib doses: deaths 0.24 (0.10-0.48); serious infections (SIs) 1.80 (1.37-2.32); herpes zoster (HZ; non-serious and serious) 3.24 (2.63-3.94); serious HZ 0.24 (0.10-0.48); opportunistic infections 0.96 (0.65-1.36); malignancies (excluding non-melanoma skin cancer [NMSC]) 0.88 (0.59-1.26); NMSC 0.71 (0.45-1.07); major adverse cardiovascular events 0.27 (0.12-0.52); deep vein thrombosis 0.06 (0.01-0.22); pulmonary embolism 0.18 (0.07-0.40); and gastrointestinal perforations 0.09 (0.02-0.27). CONCLUSIONS: Except for HZ and SIs, IRs for AESI were <1 case/100 PY. Safety was consistent with previous analyses of shorter exposure and tofacitinib's known safety profile, including real-world data. CLINICALTRIALS: GOV: NCT00787202; NCT01465763; NCT01458951; NCT01458574; NCT01470612; NCT03281304.

Etrasimod for the Treatment of Ulcerative Colitis: Analysis of Infection Events From the ELEVATE UC Clinical Program.

BACKGROUND AND AIMS: Infections are a safety concern in patients with ulcerative colitis (UC). Etrasimod is an oral, once-daily (QD), selective sphingosine 1phosphate (S1P)1,4,5 receptor modulator for the treatment of moderately to severely active UC. It leads to selective and reversible lymphocyte sequestration, and partial peripheral lymphocyte count decrease. We report infection events from the phase 3 ELEVATE program. METHODS: Proportions, incidence rates (IRs; per 100 patient-years) and descriptive analyses of all, serious, severe, herpes zoster, and opportunistic infections are reported in the Pivotal UC cohort (ELEVATE UC 52 and ELEVATE UC 12). Cox regression models evaluated potential baseline risk factors. RESULTS: In this analysis (n=787), proportions (IRs) of all infection events were similar for patients receiving etrasimod 2 mg QD (18.8% [41.1]) or placebo (17.7% [49.0]). Serious infections occurred in three (0.6%) and five (1.9%) patients receiving etrasimod and placebo, respectively. Two herpes zoster events were reported in each group (etrasimod: 0.4%; placebo: 0.8%); all localized and non-serious. One opportunistic infection event was reported in each group. No patient with an absolute lymphocyte count (ALC) <0.2 × 109/L reported serious/severe or opportunistic infections; no baseline risk factors were identified for such events. No deaths occurred. CONCLUSIONS: Patients receiving etrasimod demonstrated no increased risk of infection. The incidence of serious infections and herpes zoster was similar in each group. Among patients receiving etrasimod, no association between ALC <0.5 × 109/L and infection events was observed. Longer-term follow-up will further characterize the etrasimod safety profile.

Screening and diagnosis of cardiovascular disease using artificial intelligence-enabled cardiac magnetic resonance imaging.

Cardiac magnetic resonance imaging (CMR) is the gold standard for cardiac function assessment and plays a crucial role in diagnosing cardiovascular disease (CVD). However, its widespread application has been limited by the heavy resource burden of CMR interpretation. Here, to address this challenge, we developed and validated computerized CMR interpretation for screening and diagnosis of 11 types of CVD in 9,719 patients. We propose a two-stage paradigm consisting of noninvasive cine-based CVD screening followed by cine and late gadolinium enhancement-based diagnosis. The screening and diagnostic models achieved high performance (area under the curve of 0.988 ± 0.3% and 0.991 ± 0.0%, respectively) in both internal and external datasets. Furthermore, the diagnostic model outperformed cardiologists in diagnosing pulmonary arterial hypertension, demonstrating the ability of artificial intelligence-enabled CMR to detect previously unidentified CMR features. This proof-of-concept study holds the potential to substantially advance the efficiency and scalability of CMR interpretation, thereby improving CVD screening and diagnosis.

Personalized transcriptome signatures in a cardiomyopathy stem cell biobank.

BACKGROUND: There is growing evidence that pathogenic mutations do not fully explain hypertrophic (HCM) or dilated (DCM) cardiomyopathy phenotypes. We hypothesized that if a patient's genetic background was influencing cardiomyopathy this should be detectable as signatures in gene expression. We built a cardiomyopathy biobank resource for interrogating personalized genotype phenotype relationships in human cell lines. METHODS: We recruited 308 diseased and control patients for our cardiomyopathy stem cell biobank. We successfully reprogrammed PBMCs (peripheral blood mononuclear cells) into induced pluripotent stem cells (iPSCs) for 300 donors. These iPSCs underwent whole genome sequencing and were differentiated into cardiomyocytes for RNA-seq. In addition to annotating pathogenic variants, mutation burden in a panel of cardiomyopathy genes was assessed for correlation with echocardiogram measurements. Line-specific co-expression networks were inferred to evaluate transcriptomic subtypes. Drug treatment targeted the sarcomere, either by activation with omecamtiv mecarbil or inhibition with mavacamten, to alter contractility. RESULTS: We generated an iPSC biobank from 300 donors, which included 101 individuals with HCM and 88 with DCM. Whole genome sequencing of 299 iPSC lines identified 78 unique pathogenic or likely pathogenic mutations in the diseased lines. Notably, only DCM lines lacking a known pathogenic or likely pathogenic mutation replicated a finding in the literature for greater nonsynonymous SNV mutation burden in 102 cardiomyopathy genes to correlate with lower left ventricular ejection fraction in DCM. We analyzed RNA-sequencing data from iPSC-derived cardiomyocytes for 102 donors. Inferred personalized co-expression networks revealed two transcriptional subtypes of HCM. The first subtype exhibited concerted activation of the co-expression network, with the degree of activation reflective of the disease severity of the donor. In contrast, the second HCM subtype and the entire DCM cohort exhibited partial activation of the respective disease network, with the strength of specific gene by gene relationships dependent on the iPSC-derived cardiomyocyte line. ADCY5 was the largest hubnode in both the HCM and DCM networks and partially corrected in response to drug treatment. CONCLUSIONS: We have a established a stem cell biobank for studying cardiomyopathy. Our analysis supports the hypothesis the genetic background influences pathologic gene expression programs and support a role for ADCY5 in cardiomyopathy.

Mitigation of Stress-induced Structural Remodeling and Functional Deficiency in iPSC-CMs with PLN R9C Mutation by Promoting Autophagy.

Background: Phospholamban (PLN) is a key regulator of cardiac function connecting adrenergic signaling and calcium homeostasis. The R9C mutation of PLN is known to cause early onset dilated cardiomyopathy (DCM) and premature death, yet the detailed mechanisms underlie the pathologic remodeling process are not well defined in human cardiomyocytes. The aim of this study is to unravel the role of PLN R9C in DCM and identify potential therapeutic targets. Methods: PLN R9C knock-in (KI) and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated and comprehensively examined for their expression profile, contractile function, and cellular signaling under both baseline conditions and following functional challenges. Results: PLN R9C KI iPSC-CMs exhibited near-normal morphology and calcium handling, slightly increased contractility, and an attenuated response to ß-adrenergic activation compared to wild-type (WT) cells. However, treatment with a maturation medium (MM) has induced fundamentally different remodeling in the two groups: while it improved the structural integrity and functional performance of WT cells, the same treatment result in sarcomere disarrangement, calcium handling deficiency, and further disrupted adrenergic signaling in PLN R9C KI cells. To understand the mechanism, transcriptomic analysis showed the enrichment of protein homeostasis signaling pathways specifically in PLN R9C KI cells in response to the MM treatment and increased contractile demands. Further studies also indicated elevated ROS levels, interrupted autophagic flux, and increased pentamer PLN aggregation in functionally challenged KI cells. These results were further confirmed in patient-specific iPSC-CM models, suggesting that functional stresses exacerbate the deficiencies in PLN R9C cells through disrupting protein homeostasis. Indeed, treating stressed patient cells with autophagy-accelerating reagents, such as metformin and rapamycin, has restored autophagic flux, mitigated sarcomere disarrangement, and partially rescued ß-adrenergic signaling and cardiac function. Conclusions: PLN R9C leads to a mild increase of calcium recycling and contractility. Functional challenges further enhanced contractile and proteostasis stress, leading to autophagic overload, structural remodeling, and functional deficiencies in PLN R9C cardiomyocytes. Activation of autophagy signaling partially rescues these effects, revealing a potential therapeutic target for DCM patients with the PLN R9C mutation. Graphic abstracts: A graphic abstract is available for this article.

Efficacy and Safety of Etrasimod in Patients with Moderately to Severely Active Isolated Proctitis: Results From the Phase 3 ELEVATE UC Clinical Programme.

BACKGROUND AND AIMS: Pivotal trials in ulcerative colitis have historically excluded patients with isolated proctitis. Etrasimod is an oral, oncedaily, selective sphingosine 1phosphate1,4,5 receptor modulator for the treatment of moderately to severely active ulcerative colitis. This post hoc analysis assessed efficacy and safety of etrasimod 2 mg once daily in patients with isolated proctitis (centrally read) from the phase 3 ELEVATE UC 52 and ELEVATE UC 12 trials. METHODS: Patients, including those with isolated proctitis (<10 cm rectal involvement) who met all other inclusion criteria in ELEVATE UC 52 and ELEVATE UC 12, were randomised 2:1 to receive etrasimod or placebo. Primary, secondary and other identified efficacy endpoints and safety were assessed. RESULTS: We analysed data from 64 and 723 patients at Week 12 (both trials pooled), and 36 and 397 patients at Week 52 (ELEVATE UC 52 only) with isolated proctitis and more extensive colitis (≥10 cm rectal involvement), respectively. Patients with isolated proctitis receiving etrasimod demonstrated significant improvements versus placebo, including clinical remission rates at Weeks 12 (42.9% vs 13.6%) and 52 (44.4% vs 11.1%), endoscopic improvement (52.4% vs 22.7%) at Week 12 and bowel urgency numerical rating scale score at Week 12 (all p<0.01). Generally similar trends were observed in patients with more extensive colitis. Safety was consistent across subgroups, with no new findings. CONCLUSIONS: Etrasimod demonstrated significant improvements versus placebo in patients with isolated proctitis, and those with more extensive disease, in most efficacy endpoints at Week 12 and 52.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Mitochondrial reactive oxygen species impact human fibroblast responses to protracted γ-ray exposures.

Purpose: Continuous exposure to ionizing radiation at a low dose rate poses significant health risks to humans on deep space missions, prompting the need for mechanistic studies to identify countermeasures against its deleterious effects. Mitochondria are a major subcellular locus of radiogenic injury, and may trigger secondary cellular responses through the production of reactive oxygen species (mtROS) with broader biological implications. Methods and Materials: To determine the contribution of mtROS to radiation-induced cellular responses, we investigated the impacts of protracted γ-ray exposures (IR; 1.1 Gy delivered at 0.16 mGy/min continuously over 5 days) on mitochondrial function, gene expression, and the protein secretome of human HCA2-hTERT fibroblasts in the presence and absence of a mitochondria-specific antioxidant mitoTEMPO (MT; 5 µM). Results: IR increased fibroblast mitochondrial oxygen consumption (JO2) and H2O2 release rates (JH2O2) under energized conditions, which corresponded to higher protein expression of NADPH Oxidase (NOX) 1, NOX4, and nuclear DNA-encoded subunits of respiratory chain Complexes I and III, but depleted mtDNA transcripts encoding subunits of the same complexes. This was associated with activation of gene programs related to DNA repair, oxidative stress, and protein ubiquination, all of which were attenuated by MT treatment along with radiation-induced increases in JO2 and JH2O2. IR also increased secreted levels of interleukin-8 and Type I collagens, while decreasing Type VI collagens and enzymes that coordinate assembly and remodeling of the extracellular matrix. MT treatment attenuated many of these effects while augmenting others, revealing complex effects of mtROS in fibroblast responses to IR. Conclusion: These results implicate mtROS production in fibroblast responses to protracted radiation exposure, and suggest potentially protective effects of mitochondrial-targeted antioxidants against radiogenic tissue injury in vivo.