bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Diagnostic Performance of a Tumor Marker Gene Test to Personalize Serum CA19-9 Reference Ranges
Authors: Dbouk Mohamad , Abe Toshiya , Koi Chiho , Ando Yohei , Saba Helena , Abou Diwan Elizabeth , Macgregor-Das Anne , Blackford Amanda L. , Mocci Evelina , Beierl Katie , Dbouk Ali , He Jin , Burkhart Richard , Lennon Anne Marie , Sokoll Lori , Canto Marcia Irene. , Eshleman James R. , Goggins Michael
Published: October 2023

Abstract
Purpose: CA19-9 synthesis is influenced by common variants in the fucosyltransferase (FUT) enzymes FUT3 and FUT2. We developed a clinical test to detect FUT variants and evaluated its diagnostic performance for pancreatic ductal adenocarcinoma (PDAC). Experimental Design: A representative set of controls from the Cancer of the Pancreas Screening study was identified for each FUT functional group. Diagnostic sensitivity was determined first in a testing set of 234 PDAC cases, followed by a 134-case validation set, all of whom had undergone resection with curative intent without neoadjuvant therapy. Tumor marker gene testing was performed in the Johns Hopkins Molecular Diagnostic Laboratory. CA19-9 levels were measured in the Hopkins Clinical Chemistry lab. Receiver operating characteristic (ROC) curve analysis was used to evaluate the discriminative ability of CA19-9 alone versus with the gene test. Results: Applying the CA19-9 standard cutoff (<36U/ml) to all 716 subjects yielded a 68.8% sensitivity in the test set of cases, 67.2% in the validation set, at 91.4% specificity. Applying 99th percentile cut-offs according to each individual’s FUT group, (3, 34.9, 41.8, and 89.2, for the FUT3-null, FUT-low, FUT-intermediate, and FUT-high groups, respectively), yielded a diagnostic sensitivity for CA19-9 in the first set of cases of 66.7%, 65.7% in the validation set, at 98.9% specificity. ROC analysis for CA19-9 alone yielded an area under the curve (AUC) of 0.84; with the tumor marker gene test, AUC improved to 0.92 (p<0.001). Conclusion: Using a tumor marker gene test to personalize an individual’s CA19-9 reference range significantly improves diagnostic accuracy.
Data from Neoantigen targetability in progressive advanced melanoma
Authors: van den Bulk Jitske , Verdegaal Els M.E. , van der Ploeg Manon , Visser Marten , Nunes Joana B. , de Ru Arnoud H. , Tjokrodirijo Rayman T.N. , Ijsselsteijn Marieke E. , Janssen Natasja I. , van der Breggen Ruud , de Bruin Linda , de Kok Pita , Janssen George M.C. , Ruano Dina , Kapiteijn Ellen H.W. , van veelen Peter A. , de Miranda Noel F.C.C. , van der Burg Sjoerd H.
Published: October 2023

Abstract
Purpose: The availability of (neo)antigens and the infiltration of tumors by (neo)antigen-specific T cells are crucial factors in cancer immunotherapy. In this study, we aimed to investigate the targetability of (neo)antigens in advanced progessive melanoma and explore the potential for continued T cell-based immunotherapy. Experimental Design: We examined a cohort of eight melanoma patients who had sequential metastases resected at early and later time points. Antigen presenting capacity was assessed using immunohistochemistry and flow cytometry. T cell infiltration was quantified through multiplex immunofluorescence. Whole-exome and RNA sequencing were conducted to identify neoantigens and assess the expression of neoantigens and tumor-associated antigens. Mass spectrometry was employed to evaluate antigen presentation. Tumor recognition by autologous T cells was assessed by coculture assays with cell lines derived from the metastatic lesions. Results: We observed similar T cell infiltration in paired early and later metastatic lesions. While elements of the antigen presenting machinery were affected in some later metastatic lesions, both the early and later metastasis-derived cell lines were recognized by autologous T cells. At the genomic level, the (neo)antigen landscape was dynamic, but the (neo)antigen load was stable between paired lesions. Conclusions: Our findings indicate that subsequently isolated tumors from late-stage melanoma patients retain sufficient antigen presenting capacity, T cell infiltration, and a stable (neo)antigen load, allowing recognition of tumor cells by T cells. This indicates a continuous availability of T cell targets in metastases occurring at different time points and supports further exploration of neoantigen-specific T cell-based therapeutic approaches for advanced melanoma.
Data from Immune marker spatial distribution and clinical outcome after PD-1 blockade in mismatch repair-deficient, advanced colorectal carcinomas
Authors: Saberzadeh-Ardestani Bahar , Graham Rondell P. , McMahon Sara , Ahanonu Eze , Shi Qian , Williams Crystal , Hubbard Antony , Zhang Wenjun , Muranyi Andrea , Yan Dongyao , Jin Zhaohui , Shanmugam Kandavel , Sinicrope Frank A.
Published: October 2023

Abstract
Purpose: Targeting the PD-1/PD-L1 interaction has led to durable responses in fewer than half of patients with mismatch repair-deficient (MMR-d) advanced colorectal cancers (CRC). Immune contexture, including spatial distribution of immune cells in the tumor microenvironment, may predict immunotherapy outcome. Patients and Methods: Immune contexture and spatial distribution, including cell-to-cell distance measurements, were analyzed by multiplex immunofluorescence in primary CRCs with d-MMR (N=33) from patients treated with anti-PD-1 antibodies. By digital image analysis, density, ratio, intensity, and spatial distribution of PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFβR2, MHC-I, CD14, B2M, and pan-cytokeratin were computed. Feature selection was performed by regularized Cox regression with LASSO, and a proportional hazards model was fitted to predict progression-free survival (PFS). Results: For predicting survival among patients with MMR-d advanced CRC receiving PD-1 blockade, cell-to-cell distance measurements, but not cell densities or ratios, achieved statistical significance univariately. By multivariable feature selection, only mean number of PD-1+ cells within 10μm of a PD-L1+ cell was significantly predictive of progression-free survival (PFS). Dichotomization of this variable revealed that those with high versus low values had significantly prolonged PFS [median not reached (>83 months) vs 8.5 months (95% CI: 4.7-NR)] with a median PFS of 28.4 months for all patients [HRadj= 0.14, 95% CI: 0.04, 0.56; p=0.005]. Expression of PD-1 was observed on CD8+ T-cells; PD-L1 on CD3+ and CD8+ T-lymphocytes, macrophages (CD68+) and tumor cells. Conclusions: In d-MMR CRCs, PD-1+ to PD-L1+ receptor to ligand proximity is a potential predictive biomarker for the effectiveness of PD-1 blockade.
Data from Targeting Hedgehog Signaling with Glasdegib in Patients with Refractory Sclerotic Chronic GVHD: A Report of Two Phase I/II Trials
Authors: Rodríguez-Arbolí Eduardo , Lee Catherine J. , Caballero-Velázquez Teresa , Martínez Carmen , García-Calderón Clara , Jiménez-León María Reyes , Bermúdez-Rodríguez María Aránzazu , López-Corral Lucía , Triguero Ana , Onstad Lynn , Horwitz Mitchell E. , Sarantopoulos Stefanie , Lee Stephanie J. , Pérez-Simón José Antonio
Published: October 2023

AbstractPurpose:
Sclerotic chronic GVHD (scGVHD) is characterized by progressive skin fibrosis and frequent refractoriness to available therapies. Aberrant activation of Hedgehog signaling in dermal fibroblasts has been implicated in scGVHD. Here, we report the results of two phase I/II studies (NCT03415867, GETH-TC; NCT04111497, FHD) that evaluated glasdegib, a smoothened antagonist, as a novel therapeutic agent in refractory scGVHD.
Patients and Methods:
Adult patients with active scGVHD after ≥1 (FHD) or ≥2 (GETH-TC) lines of therapy were enrolled. Primary endpoints were dose-limiting toxicity (DLT) and MTD in the GETH-TC trial, and safety and tolerability measures in the FHD trial. Glasdegib was administered once daily in 28-day cycles. Responses were scored per 2014 NIH cGVHD criteria. Correlative studies were performed to evaluate the role of fibroblast-independent immune mechanisms on clinical activity.
Results:
Twenty (GETH-TC) and 15 (FHD) patients were recruited. Treatment-emergent grade (G) ≥2 adverse events (AE) in the GETH-TC trial included muscle cramps (85%), alopecia (50%), and dysgeusia (35%). Two patients experienced a DLT (G3 muscle cramps), and the MTD was established at 50 mg. G3 muscle cramps were the most frequently reported AE (33%) in the FHD trial. At 12-months, the skin/joint scGVHD overall response rate was 65% (all partial responses) in the GETH-TC trial and 47% (6 partial responses, 1 complete response) in the FHD cohort. No immune correlates of response were identified.
Conclusions:
Glasdegib demonstrated promising responses in patients with refractory scGVHD, but tolerability was limited by muscle cramping.
Long-Term Impact of Renin-Angiotensin System Inhibitors for Secondary Prevention in Patients with Chronic Kidney Disease Who Underwent Percutaneous Coronary Intervention
Authors: Fukase Tatsuya , Dohi Tomotaka , Nishio Ryota , Takeuchi Mitsuhiro , Takahashi Norihito , Chikata Yuichi , Endo Hirohisa , Doi Shinichiro , Nishiyama Hiroki , Okai Iwao , Iwata Hiroshi , Okazaki Shinya , Miyauchi Katsumi , Daida Hiroyuki , Minamino Tohru
Published: October 2023

Introduction: The long-term impact of renin-angiotensin system (RAS) inhibitors for secondary prevention in patients with chronic kidney disease (CKD) and coexisting coronary artery disease remains unclear. Methods: Altogether, 1,160 consecutive patients with CKD (mean age, 70 ± 9 years; 78% men) who underwent their first percutaneous coronary intervention (PCI) between 2000 and 2018 were included and analyzed. Based on their RAS inhibitor use, 674 patients (58%) were allocated to the RAS inhibitor group, and 486 patients (42%) were allocated to the non-RAS inhibitor group. This study evaluated the incidence of 3-point major adverse cardiovascular events (3P-MACE), including cardiovascular death, nonfatal acute coronary syndrome and nonfatal stroke, admission for heart failure (HF), target vessel revascularization (TVR), and all-cause death. Results: During a median follow-up duration of 7.8 years, 280 patients (24.1%) developed 3P-MACE, 134 patients (11.6%) were hospitalized for HF, 171 patients (14.7%) underwent TVR, and 348 patients (30.0%) died of any causes. The cumulative incidence rate of 3P-MACE in the RAS inhibitor group was significantly lower than in the non-RAS inhibitor group (31.7% vs. 39.0%, log-rank test, p = 0.034); however, that of admission for HF in the RAS inhibitor group was significantly higher than in the non-RAS inhibitor group (28.1% vs. 13.3%, log-rank test, p < 0.001). The subgroup of preserved ejection fraction, non-acute myocardial infarction, and non-proteinuria tended to promote the onset of HF rather than cardiovascular prevention by RAS inhibitors. Conclusion: The long-term RAS inhibitor use for patients with CKD after PCI might prevent cardiovascular events but increase the risk of HF.
Spectrum of Cerebrovascular Disease in Scleroderma: A Case Series and Systematic Review
Authors: Rosso Michela , Ramaswamy Srinath , Aharonoff David , Inanç Murat , Levine Steven R.
Published: October 2023

Introduction: Central nervous system involvement in scleroderma has traditionally been considered uncommon. Recent studies suggest that scleroderma might be associated with an increased risk of cerebrovascular disease (CBVD), independent of conventional cardiovascular risk factors. We present a case series and a systematic review to capture the spectrum of CBVD in scleroderma, through a detailed description of clinical, demographic, laboratory, and radiographical findings. Methods: In our case series, we included consecutive patients with scleroderma and CBVD seen over 35 years by our group in different hospitals in the USA. We also performed a systematic review from inception to July 2022. MEDLINE/Embase/WoS were searched for “scleroderma”, “systemic scleroderma”, “systemic sclerosis”, “cerebrovascular”, “stroke”, “cerebrovascular disorders”, “cerebrovascular disease”. Results: Fourteen patients with scleroderma and CBVD were included in our case series (mean age 48 years, 85% female). CBVDs were ischemic stroke (64%), hemorrhagic stroke (7%), venous thrombosis (7%), ischemic optic neuropathy (7%), probable ischemic stroke (14%). Of the 110 studies identified in our systematic review (45,484 patients), 82 reports with patient-level data were included for quantitative analysis (93 patients, mean age 48 years, 79% female). Despite 16 different CBVD types identified, ischemic stroke was the most common CBVD (29%), followed by vasculopathy (20%), hemorrhage (12%), vasculitis (11%), and intracranial aneurysm (11%). Conclusion: Our relatively large case series combined with a systematic review of CBVD in SCL patients shows a heterogeneous spectrum of CBVD etiology, with acute ischemic stroke being the most common in our cases and in our literature review. A complex interaction between chronic inflammation, autoimmune mechanisms, and endothelial dysfunction seems to underlie the CBVD heterogeneity in scleroderma patients. This review informs clinicians about the spectrum of CBVD related to scleroderma and raise awareness about scleroderma being a possible risk factor for early-onset CBVD.
Longitudinal Changes in Acylated versus Unacylated Ghrelin Levels May Be Involved in the Underlying Mechanisms of the Switch in Nutritional Phases in Prader-Willi Syndrome
Authors: Grootjen Lionne N. , Diene Gwenaelle , Molinas Catherine , Beauloye Véronique , Huisman T. Martin , Visser Jenny A. , Delhanty Patric J.D. , Kerkhof Gerthe F. , Tauber Maithe , Hokken-Koelega Anita C.S.
Published: October 2023

Introduction: Prader-Willi syndrome (PWS) is characterized by a switch from failure to thrive to excessive weight gain and hyperphagia in early childhood. An elevated, more unfavorable ratio between acylated and unacylated ghrelin (AG/UAG ratio) might play a role in the underlying mechanisms of this switch. We aimed to assess the evolution of the appetite-regulating hormones acylated ghrelin (AG) and unacylated ghrelin (UAG) and the AG/UAG ratio and their association with the change in eating behavior in children with PWS, compared to healthy age-matched controls. Methods: A longitudinal study was conducted in 134 children with PWS and 157 healthy controls, from the Netherlands, France, and Belgium. Levels of AG and UAG and the AG/UAG ratio were measured and nutritional phases as reported for PWS were scored. Results: The AG/UAG ratio was lower in the first years of life in PWS than in controls and started to increase from the age of 3 years, resulting in a high-normal AG/UAG ratio compared to controls. The AG levels remained stable during the different nutritional phases (p = 0.114), while the UAG levels decreased from 290 pg/mL in phase 1a to 137 pg/mL in phase 2b (p < 0.001). The AG/UAG ratio increased significantly from 0.81 in phase 2a to 1.24 in phase 2b (p = 0.012). Conclusions: The change from failure to thrive to excessive weight gain and hyperphagia in infants and children with PWS coincides with an increase in AG/UAG ratio. The increase in AG/UAG ratio occurred during phase 2a, thus before the onset of hyperphagia.
Colonic Anastomotic Leak Model in Swine
Authors: Helwa Nour , Sharma Manaswi , Vanama Manasvi Sai , Helwa Youssef , El-Falou Abdallah
Published: October 2023

Introduction: Anastomotic leaks (ALs) are serious postoperative complications. Current experimental studies designed to investigate leaks are based on acute intraoperative dehiscence of the anastomosis. Clinically, however, AL usually happens later in the postoperative course. Presented here is a clinically relevant colonic AL model in swine. Methods: Seventeen Yorkshire pigs were divided into 2 groups: the control group (n = 6) and the experimental group (n = 11). An enterotomy was performed on the descending colon and an end-to-end handsewn anastomosis was created in the groups. The proximal and distal ends of the suture were exteriorized and tied to a plastic tube. Subsequently, the suture was cut and pulled to induce breakdown of the anastomosis in the experimental group 3–4 h postoperatively. Study endpoints included behavioral changes, clinical assessment, laboratory indicators, and macroscopic indicators of leakage. Results: Leaks were successfully created in 8/11 of the experimental group animals and confirmed through exploratory relaparotomy. Seven of the experimental pigs showed complete anastomotic breakdown and one showed partial rupture. Fecal peritonitis and enteric spillage were observed macroscopically within the abdomen of the experimental pigs, confirming the presence of a leak. The remaining (3/11) experimental pigs did not experience those findings due to either a tamponade/containment by the abdominal wall or surrounding organs. Statistical significance (p < 0.05) was achieved between the experimental and control cohorts for laboratory and clinical indicators including fever, leukocytosis, and decreased blood potassium. Conclusion: This animal model generated postoperative induced leak in approximately three-quarters (8/11) of experimental pigs, allowing control over the time of leak onset to simulate clinical settings.
Cardiac Autonomic Dysfunction and Risk of Silent Myocardial Infarction Among Adults With Type 2 Diabetes
Authors: Kaze Arnaud D. , Fonarow Gregg C. , Echouffo‐Tcheugui Justin B.
Published: October 2023

Background

There is a paucity of large‐scale epidemiological studies on the link between cardiac autonomic neuropathy (CAN) and the risk of silent myocardial infarction (SMI) in type 2 diabetes. We evaluated the association between CAN and the risk of SMI in a large sample of adults with type 2 diabetes.

Methods and Results

Participants with type 2 diabetes from the ACCORD (Action to Control Cardiovascular Risk in Diabetes) study without atherosclerotic cardiovascular disease at baseline were included. CAN was ascertained using heart rate variability indices calculated from 10‐s resting electrocardiograms. The heart rate variability indices included standard deviation of all normal‐to‐normal R‐R intervals and root mean square of successive differences between normal‐to‐normal R‐R intervals. CAN was defined as both the standard deviation of all normal‐to‐normal R‐R intervals and root mean square of successive differences between normal‐to‐normal R‐R intervals less than the fifth percentile of the general population. We used Cox proportional hazards regression to generate hazard ratios (HRs) for incident SMI in relation to CAN measures. Among 4842 participants (mean age, 62.5 years; 46.6% women; 60.2% White), there were 73 incident SMI cases over a median follow‐up of 4.9 years (incidence rate 3.1 out of 1000 person‐years [95% CI, 2.5–3.9]). After adjusting for confounders, low heart rate variability was associated with a higher risk of SMI (HR, 1.67 [95% CI, 1.02–2.72] and HR, 1.56 [95% CI, 0.94–2.58] for low standard deviation of all normal‐to‐normal R‐R intervals and root mean square of successive differences between normal‐to‐normal R‐R intervals, respectively). Participants with CAN had a 1.9‐fold greater risk of SMI (HR, 1.91 [95% CI, 1.14–3.20]).

Conclusions

In a large cohort of adults with type 2 diabetes, CAN was significantly associated with an increased risk of incident SMI.
Tenecteplase, 0.4 mg/kg, in Moderate and Severe Acute Ischemic Stroke: A Pooled Analysis of NOR‐TEST and NOR‐TEST 2A
Authors: Novotny Vojtech , Kvistad Christopher Elnan , Naess Halvor , Logallo Nicola , Fromm Annette , Khanevski Andrej Netland , Thomassen Lars
Published: October 2023

Background

The optimal dose of tenecteplase in acute ischemic stroke remains to be defined. We present a pooled analysis of the 2 NOR‐TESTs (Norwegian Tenecteplase Stroke Trials) exploring the efficacy and safety of tenecteplase, 0.4 mg/kg.

Methods and Results

We retrospectively reviewed 2 PROBE (Prospective Randomized Open, Blinded End‐point) trials, NOR‐TEST and NOR‐TEST 2A. Patients were randomized to either tenecteplase, 0.4 mg/kg, or alteplase, 0.9 mg/kg. The primary end point was favorable functional outcome at 3 months (modified Rankin Scale score, 0–1) or return to baseline if prestroke modified Rankin Scale score was 2. Secondary end points included favorable functional and clinical outcome and safety data. The pooled analysis includes patients with National Institutes of Health Stroke Scale score ≥6 from both trials and an additional post hoc analysis of patients with National Institutes of Health Stroke Scale score ≤5 from NOR‐TEST. The per‐protocol analysis contains 483 patients, of whom 235 were assigned to tenecteplase and 248 were assigned to alteplase. In per‐protocol analysis, functional outcome was better in the alteplase arm with cutoff modified Rankin Scale score of 2 (odds ratio [OR], 0.52 [95% CI, 0.33–0.80];
P
=0.003) and expressed by ordinal shift analysis (OR, 1.64 [95% CI, 1.17–2.28];
P
=0.004). Mortality at 3 months was higher in the tenecteplase arm (OR, 2.48 [95% CI, 1.20–5.10];
P
=0.01). Mortality and intracranial hemorrhage rates were higher in the severe stroke group randomized to tenecteplase, whereas these rates were similar for alteplase and tenecteplase in moderate and mild stroke.

Conclusions

Tenecteplase, 0.4 mg/kg, is unsafe in moderate and severe stroke, and the risk of death and intracranial hemorrhage probably increases with stroke severity. A lower tenecteplase dose should be tested in future trials.

Registration

URL:
https://www.clinicaltrials.gov
; Unique identifiers: NCT01949948, NCT03854500.
Deep Learning Algorithms to Detect Murmurs Associated With Structural Heart Disease
Authors: Prince John , Maidens John , Kieu Spencer , Currie Caroline , Barbosa Daniel , Hitchcock Cody , Saltman Adam , Norozi Kambiz , Wiesner Philipp , Slamon Nicholas , Del Grippo Erica , Padmanabhan Deepak , Subramanian Anand , Manjunath Cholenahalli , Chorba John , Venkatraman Subramaniam
Published: October 2023

Background

The success of cardiac auscultation varies widely among medical professionals, which can lead to missed treatments for structural heart disease. Applying machine learning to cardiac auscultation could address this problem, but despite recent interest, few algorithms have been brought to clinical practice. We evaluated a novel suite of Food and Drug Administration‐cleared algorithms trained via deep learning on >15 000 heart sound recordings.

Methods and Results

We validated the algorithms on a data set of 2375 recordings from 615 unique subjects. This data set was collected in real clinical environments using commercially available digital stethoscopes, annotated by board‐certified cardiologists, and paired with echocardiograms as the gold standard. To model the algorithm in clinical practice, we compared its performance against 10 clinicians on a subset of the validation database. Our algorithm reliably detected structural murmurs with a sensitivity of 85.6% and specificity of 84.4%. When limiting the analysis to clearly audible murmurs in adults, performance improved to a sensitivity of 97.9% and specificity of 90.6%. The algorithm also reported timing within the cardiac cycle, differentiating between systolic and diastolic murmurs. Despite optimizing acoustics for the clinicians, the algorithm substantially outperformed the clinicians (average clinician accuracy, 77.9%; algorithm accuracy, 84.7%.)

Conclusions

The algorithms accurately identified murmurs associated with structural heart disease. Our results illustrate a marked contrast between the consistency of the algorithm and the substantial interobserver variability of clinicians. Our results suggest that adopting machine learning algorithms into clinical practice could improve the detection of structural heart disease to facilitate patient care.
Abstract EC.01: Endocardial Hdac3 Promotes Myocardial Trabeculation Through A MicroRNA Pathway
Authors: Jang Jihyun , Li Deqiang
Published: October 2023

A key process during early heart development is myocardial trabeculation, which is the formation of bundles of myocardium extending into the chamber. The failure of trabeculation often contributes to many forms of congenital heart defects. Proper trabeculation requires coordination among multiple cell types and signaling communication. For instance, growth signals released from the endocardium are required for trabecular myocardial growth and maturation. However, it is poorly understood how these signaling cascades are initiated and regulated in the endocardium. Recently, our lab demonstrated that epicardial histone deacetylase 3 (HDAC3) is required for myocardial compaction. In this current study, we found that the genetic deletion of Histone deacetylase 3 (Hdac3) in the cardiac endothelial cells in mice resulted in early embryo lethality presenting as a hypotrabeculation cardiac phenotype, suggesting that endocardial Hdac3 deletion disrupted critical signals from the endocardium for trabecular myocardium growth. When cultured with supernatant from Hdac3 knockout (KO) mouse cardiac endothelial cells (MCECs), wild-type (WT) mouse embryonic cardiomyocytes also showed decreased proliferation. Single cell RNA-sequencing identified that transforming growth factor ß3 (Tgfß3) along with their extracellular matrix (ECM) downstream targets was significantly downregulated in Hdac3 deficient endocardial cells. Interestingly, chemical inhibition of deacetylase catalytic activity alone resulted in significant downregulation of Tgfß3 in cultured Hdac3 KO MCECs, suggesting that HDAC3 induces Tgfß3 expression in an enzymatic dependent manner. Mechanistically, we identified that miR-129 was significantly upregulated in Hdac3 KO MCECs and Hdac3 endocardial KO hearts. Overexpression of miR-129 repressed Tgfß3 expression in WT MCECs, whereas knockdown of miR-129 restored Tgfß3 expression in Hdac3 KO MCECs. Our findings reveal a critical signaling pathway in which endocardial HDAC3 promotes trabecular myocardium growth by stimulating Tgfß through repressing miR-129, providing novel insights into elucidating the etiology of congenital heart defects and conceptual strategies to promote myocardial regeneration.
Abstract GS.103: Human Cardiomyopathy Disease Variant In Formin Homology 2 Domain Containing 3 (FHOD3) Protein Revealed Novel Early Autophagy Dysregulation And Mitochondrial Dysfunction
Authors: Boukhalfa Asma , Powers Sarah N , Martin Gregory , Blanton Robert M , Huggins Gordon S , Chen Howard H
Published: October 2023

Introduction:
The FHOD3-V1151I variant has been identified by multiple Genome Wide Association Studies to be associated with hypertrophic cardiomyopathy (HCM). FHOD3 regulates actin filament formation which is central to key cardiomyocyte processes including autophagy. The precise role of autophagy in cardiomyopathy is an emerging area of research but poorly characterized. We hypothesize that V1151I may perturb FHOD3-dependent cardiac autophagy regulation.

Methods:
The homologous V1151I variant was introduced into mice using CRISPR. Cardiac function was studied by echocardiography, cardiac mitochondrial function measured by Seahorse (Agilent) and oil red O staining of lipid droplets in cardiac sections. Cultured H9C2 cardiomyoblasts were transfected with FHOD3-V1151V (WT) or V1151I (variant) expression plasmids and immunoprecipitation and immunofluorescence were performed to assess protein interaction and localization. Phosphatidylinositol 3-phosphate (PI3P, Echelon) was used for the rescue experiments.

Results:
Heart tissue from FHOD3-V1151I variant mice (n=7) showed a 5-fold (p<0.0001) increase in lipid droplet accumulation and impaired mitochondrial respiration and ATP production (p<0.0001), while cardiac structure and function assessed by echocardiography was similar to V1151V WT littermate control hearts (n=7). A bioinformatics analysis predicted FHOD3 interaction with PFN4 which we confirmed by co-immunoprecipitation in addition to ATG16, a regulator of autophagic vesicle membrane biogenesis. The FHOD3-PFN4-ATG16 interaction was however lost in FHOD3-V1151I hearts. Immunofluorescence of H9C2 cells expressing FHOD3-V1151I revealed that the variant disrupts interaction of LC3 with PI3P, a hallmark of early autophagy. The disrupted LC3-PI3P interaction seen with the V1151I variant phenocopies cells treated with early autophagy inhibitors 3-MA or spautin, but not late autophagy inhibitor chloroquine. Furthermore, exogenous PI3P restored the molecular interaction and autophagy dysregulation.

Conclusions:
We demonstrate for the first time that early autophagy dysregulation underlies energetic defects observed in cardiomyopathy, and is reversible, thus providing a novel potential therapeutic target.
Abstract P1003: Expression Of Circulating Atherosclerosis-associated Micrornas In Pre- And Post-menopausal Obese Women: Response To Physical Exercise
Authors: Faffe Debora S , Costa-dos-Santos Diego , Cabral Bianca C , Bottaro Thayane , Silva Rosane , Urmenyi Turan P
Published: October 2023

Atherosclerosis is the basic mechanism of cardiovascular disease, a major cause of mortality. It involves the interaction of environmental factors, such as obesity, and genetics, while exercise is a major risk modifier. Despite its higher prevalence in men, women have a worse outcome; however, they are underrepresented in clinical trials. MicroRNAs (miRs) are gene expression regulators of special interest as potential risk markers for complex diseases. We investigated the effect of physical exercise on the expression of circulating miRs in obese pre- and post-menopausal women. For this purpose, we analyzed serum from six women before and after performing a physical exercise protocol (3 x/week for 16 weeks). Samples were selected from women with extreme responses to the exercise protocol. The miRs were extracted using miRNeasy Serum/Plasma Kit and sequenced by mass sequencing on the Ion Torrent PGM platform. Data were analyzed using the
CLC Genomics Workbench
software. The differently expressed miRs were submitted to
in silico
analysis to determine the computational prediction of gene targets with the Mirnet tool (version 2.0). Then, the gene ontology of predicted target genes was verified using EnrichR. Altogether, five miRs with increased expression were identified for the menopausal group compared to the non-menopausal group before physical exercise (miR-199a, -222, -186, -4454, and 126). In the non-menopausal group, we identified one miR with increased expression (miR-122) and 4 with reduced expression (miR-29c, Let-7b, -339, and -98) after the exercise protocol. The menopause group showed an increase of 5 miRs (miR-15b, -19a, -107, -103a, 17) and a reduction of 4 (miR-423, -148a, -Let-7i, and -338) induced by exercise. Gene ontology analysis identified several pathways in the atherosclerosis process modulated by physical exercise. Our results showed that exercise induces the expression of circulating miRs that potentially regulates target genes involved in atherosclerosis physiopathology, inflammatory response, oxidative stress, and obesity. The pathways modulated by exercise differ among pre- and post-menopausal obese women, thus potentially impacting the approach of atherosclerosis in women.
Abstract P1009: Investigating The Effect Of Patient Missense Variants In
CHD7
Using An Induced Pluripotent Stem Cell Model
Authors: Sharma Ananya , Zhang Qi , Morton Sarah U , Seidman Jonathan G , Seidman Christine E
Published: October 2023

Congenital heart disease (CHD) is a phenotypically diverse and genetically heterogenous disease. While monogenic causes of familial CHD and damaging
de novo
variants are known contributors to CHD risk, novel variants of uncertain significance (VUS) detected among CHD probands remain under characterized. In particular, the effect of missense variants in
CHD7
(chromodomain helicase DNA binding protein 7), remain poorly explored.
CHD7
is an established CHD gene with variants associated with CHARGE syndrome (Coloboma, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital/urinary abnormalities, and Ear abnormalities) and incomplete syndromic phenotypes. We hypothesized that some
CHD7
missense variants in genetically engineered human-induced pluripotent stem cells (iPSCs) perturb essential cardiac developmental pathways.

CHD7
is highly expressed in iPSCs and during differentiation to iPSC-cardiomyocytes. Out of seventy rare patient missense variants in
CHD7
reported by the Pediatric Cardiac Genetics Consortium (PCGC), six were chosen for genomic editing. To investigate the functional effects of missense variants in
CHD7,
we first predicted pathogenicity using AlphaFold, an algorithm which models variant impact on the three-dimensional structure of proteins. We then introduced six patient missense variants (Q161R, R286G, I1500V, T262A, T894A, L2299P) into an iPSC line using adenine base editors. Heterozygous and homozygous loss-of-function
CHD7
lines were generated for comparison. RNA expression profiles of edited iPSCs were analyzed using single cell gene expression with multiplexing to assess the impact of the missense variants on transcriptional profiles. Bulk RNA sequencing was performed in parallel to better characterize transcriptional changes. These results further our understanding of how
CHD7
missense variants may contribute to CHD risk.

In the future, these approaches could be applied to a broader list of genes, as rare patient missense variants in definitive and candidate CHD genes require further investigation to determine pathogenicity. Developing iPSC models to investigate functional effects of missense variants can aid in understanding the complex genetic architecture of CHD.
Abstract P1014: Renal Autocrine Npff, Via Its Local Receptors, Regulates Blood Pressure Through Its Interaction With The Renal Dopaminergic System
Authors: Amatya Bibhas , Villar Van , Asico Laureano D , Moore Shaun , Polzin Jacob , Armando Ines , Felder Robin A , Jose Pedro A , Lee Hewang
Published: October 2023

Neuropeptide FF (NPFF), a hormone originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the role of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, is unknown. In this study, we found that NPFF (
NPFF
,
Npff
), NPFF-R1(
NPFFR1
,
Npffr1
), and NPFF-R2(
NPFFR2
,
Npffr2
) mRNAs and proteins were expressed in both mouse and human renal proximal tubules (RPTs), determined by RT-PCR, RNAScope, immunoblotting, and immunofluorescence microscopy. NPFF-R2, compared with NPFF-R1, was predominantly expressed in RPTs and RPT cells (RPTCs). In human and mouse RPTCs, NPFF decreased cAMP production in a concentration- and time-dependent manner, consistent with its receptors’ linkage to the inhibitory G protein, Gαi. The D
1
R, but not D
5
R, colocalized with NPFF-R2 in human RPTCs and human kidney tissue sections. The colocalization between D
1
R and NPFF-R2 was decreased by the treatment with NPFF (100 nM, 30 min) in mouse RPTCs. Consistent with these findings, D
1
R co-immunoprecipitated with NPFF-R2 in human RPTCs. Fenoldopam, a D1-like receptor agonist, increased the cellular cAMP production and intracellular Na
+
accumulation, which were attenuated by NPFF (100 nM, 30 min), indicating an antagonistic interaction between NPFF and D1-like receptors. Moreover, acute and chronic administration of NPFF (10 μg in 100 μL, 0.5 μL/hr) in C57Bl/6 mice increased their blood pressures (from 96.4±3 to 114±5 mmHg, n=4) that were blocked by RF9 (10 μg in 100 μL, 0.5 μL/hr), an antagonist of both NPFF-R1 and NPFF-R2. NPFF also decreased urinary sodium excretion (from 0.36±0.06 to 0.28±0.05 mEq/day, n=4). In summary, NPFF by its negative interaction with the renal dopaminergic system regulates sodium excretion and blood pressure.
Abstract P1021: Estimating Cardiac Cell Composition In Gtex With Single-nucleus Reference Profiles.
Authors: Gural Brian , Rau Christoph
Published: October 2023

Heart failure (HF) is a heterogeneous condition defined by an inability of the heart to meet the circulatory needs of the body. HF is the result of a maladaptive response to chronic stressors during which the heart undergoes structural and cellular remodeling. For example, cardiomyocytes undergo apoptosis after chronic overstimulation while fibroblasts proliferate to fill gaps left by the loss of larger cells. Although changes to the cellular makeup of the heart are known to mediate HF pathology, estimates of basal cardiac cellular composition vary widely. Moreover, despite our understanding of HF as a complex condition that is influenced by environmental and genetic factors, most studies on heart composition only examine small groups of individuals or inbred models. Recent efforts have used single-nucleus RNAseq (snRNAseq) to tally nuclear proportions as a proxy for cellular proportions. However, cellular dissociation biases for certain cell types and skews the resulting estimates. Furthermore, snRNAseq studies are expensive, making them infeasible and underpowered for population-scale analyses. To avoid these issues, we developed a method to estimate cellular composition of bulk RNAseq using cell-type-specific references defined in snRNAseq. While most deconvolution tools are designed for single-cell RNAseq inputs, our method considers the technical and biological differences of snRNAseq. It also allows for the inclusion of additional reference datasets, utilizes simulated controls, and leverages an existing deconvolution tool, MuSiC. We apply our method to the Genome-Tissue-Expression project (GTEx), a multi-tissue database with bulk RNAseq from hundreds of subjects and snRNAseq from a small subset of those subjects. We estimated left ventricular cellular composition in 386 individuals, revealing considerable heterogeneity. We found significant correlations between subject phenotypes and discrete cell populations. This analysis demonstrates our ability to leverage existing, public datasets to accurately map the understudied variability of cardiac cell type variation across human populations. We now look to incorporate genomic datasets to find genetic markers that link composition estimates to population-level variation.
Abstract P1027: Confocal And Mass Spectrometry-based Investigation Of REEP5 Depletion By AAV9 In The Mouse Heart
Authors: Di Paola Michelle , Reitz Cristine , Kuzmanov Uros , Jia Kateleen , Teng Allen , Gramolini Anthony
Published: October 2023

The sarco(endo)plasmic reticulum (SR/ER) is an essential regulator of many key cellular processes, especially those that play a role in the development and progression of cardiac disease. However, many aspects of its structural organization remain poorly defined. Receptor Expression Enhancing Protein 5 (REEP5) is a cardiac enriched SR/ER membrane protein, which regulates organization of the highly differentiated SR/ER network and responses to stress. In zebrafish models, genetic knock-out of
reep5
results in cardiac functional defects and reduced heart rate. Within the cardiomyocyte, depletion of
Reep5
in vitro
results in decreased muscle cell contraction, disrupted Ca
2+
signaling and SR/ER luminal vacuolization. For these studies,
in
vivo
cardiac knock-down of
Reep5
in the mouse was achieved using recombinant adeno-associated virus serotype 9 (rAAV9)-mediated gene delivery. Cardiac tissues or isolated cardiomyocytes were harvested at 7 days through to 4 weeks following knock-down, for biochemical and functional assessments. We observed that the largest significant change in REEP5 expression occurred 4 weeks post-rAAV9 injection, correlating to a 78% knock-down, observed by immunoblotting (unpaired t-test, p<0.0001, n=6-8). 5 weeks following knock-down, mice developed lethal cardiac dysfunction. To assess the biochemical changes induced by
Reep5
knock-down, we have established an organelle-specific cardiac proteomic profile of the microsome, mitochondria and cytosol, using subcellular fractionation and mass spectrometry (nLC-ESI-MS-HCD-MS). Coupled with high resolution confocal microscopy and 3D mapping techniques, we have examined localization and expression patterns of key SR/ER and mitochondrial proteins, that have altered expression following knock-down of
Reep5
at the myocyte-level. These findings provide a detailed understanding of the role that REEP5 plays in maintaining ER homeostasis, SR/ER structure, and general organelle integrity. By identifying the mechanistic significance of REEP5 expression in the heart, we can work to delineate underappreciated pathways in cardiac muscle development.
Abstract P1030: Alpha Kinase 3 Signaling At The M-band Regulates Sarcomeric Protein Quality Networks
Authors: McNamara James W , Parker Benjamin , Voges Holly , Mehdiabadi Neda , Bolk Francesca , Ahmad Feroz , Zech Antonia , Watt Kevin , Porrello Enzo R , Ellliott David
Published: October 2023

Objectives:
Striated muscle contraction is driven by molecular machinery composed of specialized cytoskeletal proteins, called the sarcomere. Pathogenic variants in genes that encode these sarcomeric proteins trigger a heart disease called cardiomyopathy, which causes abnormal growth and function of the heart muscle and can cause sudden death. However, there is a growing appreciation that genetic mutations in genes not encoding the traditional sarcomeric proteins also substantially contribute to cardiomyopathy. One such gene is
ALPK3
, encoding an atypical alpha kinase. Despite its strong link to cardiomyopathy, there have been no studies to define its molecular function. Therefore, the objective of our study was to understand the genetic basis of
ALPK3-
induced cardiomyopathy and define its molecular function in health and disease.

Methods & Results:
We created a suite of gene-edited human pluripotent stem cells (hPSCs) carrying endogenous fusion proteins, loss of function mutations, or disease-causing patient variants, and differentiated these hPSC lines into human ventricular muscle cells. Using reporter hPSCs, we found that ALPK3 is localized to the M-band of the cardiac sarcomere, in contrast to its posited nuclear localization. ALPK3 deficiency impaired muscle cell function and sarcomere organization in both hPSC cardiac organoids and mice harboring a pathogenic
ALPK3
variant. We performed multiomic analysis on ALPK3-deficient hPSC cardiomyocytes to define the function of ALPK3 and observed consistent enrichment of sarcomere and protein quality control pathways. Interactomics demonstrated that ALPK3 links the cytoskeleton to protein surveillance networks at the M-Band. Notably, the ubiquitin-binding protein SQSTM1 bound ALPK3, could be phosphorylated by ALPK3, and was mislocalized from the sarcomere in three independent
ALPK3
patient lines.

Conclusions:
Together, our data demonstrate that ALPK3 regulates the link between the sarcomere and protein surveillance system and that its deficiency impairs cardiac function via this mechanism. This work highlights a potential new pathway to target for therapy development and treatment of patients with cardiomyopathy.
Abstract P1032: Substrate Optimization For Patients With Melas And Leigh Syndrome Using Induced Pluripotent Stem Cells-derived Cardiomyocytes
Authors: Yamamoto Taylor H , Huang Constance C , Cutright Ava K , Dinchman Kenan C , Cohen Bruce H , Chilian William M , Kang Patrick T
Published: October 2023

Mitochondrial DNA (mtDNA) mutations increase the risk of health issues with overlapping clinical phenotypes. We propose that patient-derived induced pluripotent stem cells (iPSCs) can serve as surrogates for studying mitochondrial diseases, provided a standardized protocol is used to mitigate effects of mtDNA heteroplasmy. Accordingly, we reprogrammed peripheral blood mononuclear cells (CytoTune Kit) from mtDNA disease patients with epilepsy enrolled in clinical trials. After culturing the reprogrammed cells, six iPSC cell colonies were selected (Alkaline Phosphatase Live Stain) from each donor, pooled, and sorted by SSEA4+ marker at passage 6. Pluripotency was confirmed using OCT4 immuno-staining and RT-PCR for OCT4, NANOG, and SOX2 at passage 10. Because two of the patients (m.3243A>G MT-TL1, MELAS, and m.T8993G MT-ATP6, Leigh Syndrome) showed signs of improvement on drug therapy, we differentiated their iPSCs into cardiomyocytes (iCMs) and characterized them using immuno-staining (cTnT). We then screened the substrate utilization preferences of the iCMs using Phenotype MicroArray Mammalian assays (Biolog). The MELAS-iCMs failed to metabolize lactic acid, L-ornithine, and branched-chain amino acids, but performed better than control iCMs in the presence of arginine and glycerol phosphate. In contrast, LS-iCMs exhibited distinctly different substrate preferences and thrived in the presence of acetoacetic acid and α-keto-butyric acid with reduced oxidative stress (AmplexRed). The failure of MELAS-iCMs to survive in the presence of only lactic acid recapitulates a characteristic feature of MELAS, underscoring the potential of iPSCs for disease modeling. Our findings suggest that diverse outcomes to nutritional and pharmaceutical interventions must be tailored to each patient. To validate the pharmacological efficacy of iCMs, we will continuously evaluate their response in patients who are refractory to treatment.
Abstract P1040: An Engineered Human Cardiac Tissue Model Reveals The Potential Contribution Of Systemic Lupus Erythematosus Autoantibodies To Myocardial Injury
Authors: Nash Trevor R , Fleischer Sharon , Tamargo Manuel A , Lock Roberta I , Morsink Margaretha , Li Vanessa , Lamberti Morgan J , Graney Pamela L , Liberman Martin , Kim Youngbin , Zhuang Richard Z , Whitehead Jaron , Friedman Richard A , Soni Rajesh K , Geraldino-Pardilla Laura , Winchester Robert , Vunjak-Novakovic Gordana
Published: October 2023

A considerable number of patients with systemic lupus erythematosus (SLE) develop myocardial injury. SLE patients have highly heterogenous populations of autoantibodies that may contribute to cardiac disease pathogenesis and heterogeneity; however, little is known about their direct role in myocardial injury. Here, we engineered a model of matured human cardiac tissue and used it to study SLE induced myocardial injury. To this end, we cultured tissues with immunoglobulin G (IgG) from SLE patients with and without myocardial injury. We found that IgG binding levels differed between patient groups and correlated with measures of myocardial inflammation and systolic function. Interestingly, IgG from a subset of patients with systolic dysfunction exerted functional effects on our model system in the absence of immune cells, suggesting a direct contribution to myocardial injury. This was demonstrated by altered calcium handling and cellular composition, impaired cellular respiration, and distinctive patterns of gene expression determined by RNA sequencing. We further harnessed this model to identify potential disease-contributing autoantibodies. We propose that this model can be used to improve risk stratification and therapeutic strategies for SLE patients with myocardial injury.