bluuebunny/crossref_metadata_2025 huggingface dataset:
Preoperative H. pylori Eradication Therapy Facilitates Precise Delineation in Early Gastric Cancer with Current H. pylori Infection
Authors: Yan Zhiyu , Zou Long , Wang Qiang , Zhang Shengyu , Jiao Yuhao , Xiong Dingkun , Jiang Qingwei , Guo Tao , Feng Yunlu , Wu Dongsheng , Lai Yamin , Yan Xuemin , Xu Tao , Fang Weigang , Wu Xi , Zhou Weixun , Yang Aiming
Published: October 2023

Introduction: Early gastric cancer with current Helicobacter pylori infection (HpC-EGC) is common, but it is still unclear whether H. pylori eradication therapy (Hp-ET) or endoscopic submucosal dissection (ESD) should be performed first. We evaluated Hp-ETs short-term effects on horizontal boundary delineations of HpC-EGC in ESD. Methods: Prospectively enrolled HpC-EGC patients were randomly assigned to eradication or control groups. Operation scopes of HpC-EGC lesions were delineated with marking dots at 5 mm out of the endoscopic demarcation line by an independent endoscopist, unaware of eradication status, before formal circumferential incision. As representatives, precise delineation rate, the shortest distance of all marking dots to the pathological demarcation line in all slices of one intact resected specimen (Dmin), and negative marking dot specimen rate were examined. Results: Twenty-three HpC-EGC patients (25 lesions) were allocated to eradication group and 26 patients (27 lesions) were allocated to the control group with similar eradication success rates and all were differentiated type. With improving background mucosa inflammation after Hp-ET and similar gastritis-like epithelium rates, 10 lesions (40.0%) in the eradication group were of precise delineation compared to control group with 2 lesions (7.4%) (relative risk = 5.40, 95% CI 1.31–22.28). Dmin of eradication and control groups were 4.17 ± 2.52 mm and 2.67 ± 2.30 mm (p = 0.029), accompanied by 4 (14.8%) and none (0.0%) specimens that exhibited positive marking dots (p = 0.11), respectively. Conclusion: For HpC-EGC patients, administrating eradication medication before ESD is beneficial for the precise delineation of lesions and reducing the risk of positive horizontal resection margins.
Low Lean Tissue Index and Muscle Strength Combined with High Fat Tissue Index Are Associated with Cardiovascular Disease-Related Hospitalisation in Patients on Haemodialysis
Authors: Tian Maolu , Lan Qin , Yuan Jing , He Pinghong , Yu Fangfang , Long Changzhu , Zha Yan
Published: October 2023

Introduction: The combined clinical impact of muscle mass, muscle function, and adipose mass on hospitalisation events, especially those that have exact causes, such as cardiovascular diseases (CVDs), had been rarely studied in patients on haemodialysis (HD). This study aimed to determine the influence of lean tissue index (LTI), fat tissue index (FTI), and hand grip strength (HGS) on the risk of CVD-related hospitalisation in patients undergoing chronic HD. Methods: This multi-centre observational study enrolled a total of 2,041 clinically stable patients aged >18 years and who had undergone HD for at least 3 months at 17 HD units in 2019. The follow-up period was up to 2 years. LTI and FTI were assessed using a body composition monitoring machine, and HGS was measured by a CAMRY® dynamometer. Cox regression models were fit to estimate the associations of body composition and HGS with CVD-related hospitalisation risk. Results: During a mean follow-up of 22.6 months, CVD-related hospitalisation occurred in 492 patients. Compared with the non-CVD group, patients with CVD-related hospitalisation were older; had lower diastolic blood pressure; were more likely to have a history of diabetes; had worse activity status scores and lower levels of LTI, HGS, serum uric acid, and serum creatinine; and had higher FTI levels, body mass index, and extracellular water/intracellular water ratio. In the Cox regression models, low LTI and high FTI were independently associated with CVD-related hospitalisation in both men and women. In men, low HGS was an independent risk factor for CVD-related hospitalisation. When patients were further stratified into four distinct groups according to the sex-specific median values of LTI and FTI, the combination of low LTI and high FTI was an independent risk factor for CVD-related hospitalization (hazard ratio [HR] = 1.79 in men, 95% confidence interval 1.26–2.55; HR = 2.48 in women, 95% confidence interval 1.66–3.71; reference: high LTI/low FTI group). Conclusions: Among patients on chronic HD, low LTI, and high FTI were associated with CVD-related hospitalisation in men and women, whereas HGS was an independent risk factor for CVD-related hospitalisation in men but not in women. Combining low LTI and high FTI increased the association with hospitalisation risk and was an independent predictor of CVD-related hospitalisation.
Comparative Discrimination of Life’s Simple 7 and Life’s Essential 8 to Stratify Cardiovascular Risk: Is the Added Complexity Worth It?
Authors: Howard George , Cushman Mary , Blair Jessica , Wilson Nicole R. , Yuan Ya , Safford Monika M. , Levitan Emily B. , Judd Suzanne E. , Howard Virginia J.
Published: October 2023

BACKGROUND:

Life’s Simple 7 (LS7) is an easily calculated and interpreted metric of cardiovascular health based on 7 domains: smoking, diet, physical activity, body mass index, blood pressure, cholesterol, and fasting glucose. The Life’s Essential 8 (LE8) metric was subsequently introduced, adding sleep metrics and revisions of the previous 7 domains. Although calculating LE8 requires additional information, we hypothesized that it would be a more reliable index of cardiovascular health.

METHODS:

Both the LS7 and LE8 metrics yield scores with higher values indicating lower risk. These were calculated among 11 609 Black and White participants free of baseline cardiovascular disease (CVD) in the Reasons for Geographic and Racial Differences in Stroke study, enrolled in 2003 to 2007, and followed for a median of 13 years. Differences in 10-year risk of incident CVD (coronary heart disease or stroke) were calculated as a function LS7, and LE8 scores were calculated using Kaplan-Meier and proportional hazards analyses. Differences in incident CVD discrimination were quantified by difference in the c-statistic.

RESULTS:

For both LS7 and LE8, the 10-year risk was approximately 5% for participants around the 99th percentile of scores, and a 4× higher 20% risk for participants around the first percentile. Comparing LS7 to LE8, 10-year risk was nearly identical for individuals at the same relative position in score distribution. For example, the “cluster” of 2013 participants with an LS7 score of 7 was at the 35.8th percentile in distribution of LS7 scores, and had an estimated 10-year CVD risk of 8.4% (95% CI, 7.2%–9.8%). In a similar location in the LE8 distribution, the 1457 participants with an LE8 score of 60±2.5 at the 39.4th percentile of LE8 scores had a 10-year risk of CVD of 8.5% (95% CI, 7.1%–10.1%), similar to the cluster defined by LS7. The age-race-sex adjusted c-statistic of the LS7 model was 0.691 (95% CI, 0.667–0.705), and 0.695 for LE8 (95% CI, 0.681–0.709) (
P
for difference, 0.12).

CONCLUSIONS:

Both LS7 and LE8 were associated with incident CVD, with discrimination of the 2 indices practically indistinguishable. As a simpler metric, LS7 may be favored for use by the general population and clinicians.
Abstract EC.04: The Effects Of Cardiac Troponin Activator CK-358 On Cardiac Contractility In A Mouse Model Of Genetic Dilated Cardiomyopathy
Authors: Neys Stephanie , Kooiker Kristina , Martinson Amy , Hwee Darren , Hartman James , Murphy Anne N , Morgan Bradley , Davis Jennifer , Malik Fady , Moussavi-Harami Farid
Published: October 2023

Genetic dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. However, most treatments do not address the underlying contractile deficit caused by the disease. In an effort to create more targeted DCM therapeutics, novel small molecule drugs known as sarcomeric modulators have been of recent interest. In a recent study, a cardiac troponin activator was shown to improve cardiac muscle contractility without adversely affecting energetics or diastolic function. Here, we aim to elucidate the mechanism by which the cardiac troponin activator CK-3827358 (CK-358) is able to recover contractile function in a mouse model of DCM with a mutation at residue 61 of cardiac troponin C (cTnC I61Q). We measured steady state force and
k
tr
, a measure of crossbridge cycling kinetics, in demembranated ventricular tissue from control and cTnC I61Q mice in solutions with increasing concentrations of calcium, ranging from pCa 9 to pCa 4. The cTnC I61Q mutation significantly decreased calcium sensitivity versus control (pCa
50
: 5.48 ± 0.06 vs 5.75 ± 0.01). Compared to controls, CK-358 (1 μM) significantly increased calcium sensitivity (pCa
50
) in tissue from both cTnC I61Q and control mice (6.03 ± 0.06 and 6.50 ± 0.18, P=0.005) without any change in maximum force. CK-358 also significantly increased
k
tr
, possibly suggesting activation of thin filaments. Next, we used a fluorescently labeled human cardiac troponin C (HcTnC) conjugated to N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) combined with subunits T and I to make a full HcTn complex. We performed calcium titrations with the HcTn-IANBD and saw no change in fluorescence in the control protein with and without CK-358. Finally, we performed HcTnC titrations with HcTnI and saw no change in fluorescence at maximum, sub-maximum, and minimum calcium concentrations. Overall, we demonstrate that cardiac troponin activator CK-358 did not alter calcium binding to HcTnC or its interaction with HcTnI. However, it restores the calcium sensitivity of cTnC I61Q DCM ventricular sarcomeres. Future studies will examine the effects of CK-358 on calcium binding in decorated thin filaments and measure activation using stopped flow spectroscopy in this model of DCM.
Abstract EC.08: Novel Candidate Variant In The Gene Coding For Receptor Tyrosine-protein Kinase ErbB-2 Is Associated With Left Ventricular Outflow Tract Obstruction Defects In Human
Authors: Ampuja Minna , Selenius Sabina , Paatero Ilkka , Chowdhury Iftekhar , Villman Jenna , Broberg Martin , Ranta Amanda , Ojala Tiina , Varjosalo Markku , Kivelä Riikka , Helle Emmi
Published: October 2023

Congenital heart defects (CHD) are the most common congenital malformations. Both genetic and environmental factors, such as blood flow during development, are known to contribute to disease development. The genetic predisposition is likely in most cases oligogenic, and currently, robust evidence for few predisposing genes for monogenic forms exist. Here we show evidence for a rare variant in the ERBB2 gene as potentially causal for CHD. ERBB2 is known to be essential for cardiac development in animal models, but so far human data are lacking. We studied a Finnish cohort of left ventricular outflow tract obstruction (LVOTO) patients and their families and identified a variant in ERBB2 (R569C) that segregated with disease in three unrelated families with multiple affected members. We performed functional studies, and show that the mutated receptor is functional, as NRG1 stimulation phosphorylates the receptor. However, a protein-protein interaction assay showed that the mutated receptor is mainly located in ER/mitochondria, whereas the wild type ERBB2 is located at the plasma membrane. This was confirmed by a flow cytometry analysis showing decreased receptor presence in the plasma membrane of patient derived cells. Zebrafish embryos expressing the R599C mutant allele demonstrated altered myocardium structure and reduced contractility compared to the wild-type allele or a mock control. Finally, we differentiated induced pluripotent stem cells (iPSCs) from a patient with the variant into cardiomyocytes. Single-cell RNA sequencing demonstrated reduced expression of genes related to heart development, muscle contraction and response to oxidative stress in patient cells compared to healthy controls. As endothelial cells (EC) are important during cardiac development, we also differentiated patient and healthy control iPSCs into ECs. Transcriptomic analysis showed significant differences in the expression of angiogenesis and vasculature development related genes in the patient cells. In summary, ERBB2 R599C variant segregates with CHD patients in three unrelated families with LVOTO defects. The variant influences location of the protein and causes compromised heart function in zebrafish embryos. We propose ERBB2 as a new candidate gene for CHD.
Abstract GS.06: The Fibrogenic Transcription Factor Sox9 Controls Mesenchymal Activation Of Endothelial Cells And Drives Fibrotic Disease
Authors: Trogisch Felix A , Abouissa Aya , Keles Merve , Birke Anne , Fuhrmann Manuela , Dittrich Gesine M , Cordero Julio , El Sherbiny Adel , Martin Garrido Abel , Grein Steve , Nicin Luka , Bibli Sophia-Iris , Airik Rannar , Kispert Andreas , Kist Ralf , Quanchao Sun , Kürschner Sina W , Winkler Manuel , Gretz Norbert , Mogler Carolin , Korff Thomas , Koch Philipp-Sebastian , Dimmeler Stefanie , Dobreva Gergana , Heineke Joerg
Published: October 2023

Introduction:
Endothelial cells (ECs) undergo mesenchymal activation (EndoMA) during the onset of fibrotic cardiac remodeling. The contribution of EndoMA to disease progression, matrix deposition and upstream master regulators, however, remain elusive.

Objective:
Aim of the study was to investigate the impact of the fibrogenic transcription factor Sox9 in ECs on the development of fibrotic disease.

Methods & Results:
Following both transverse aortic contriction (TAC) and administration of high fat diet (HFD) with NOS inhibitor L-NAME in mice, as well as in human diseased hearts, we found a significant induction of
Sox9
in cardiac ECs. EC-specific overexpression of Sox9 in transgenic mice induced fibrotic and hypertrophic remodeling of heart, lung, liver and spleen, accompanied by left-ventricular diastolic followed by systolic dysfunction. Both bulk and single-cell (SC) RNA sequencing (seq) of isolated ECs as well as immunofluorescence staining showed that ECs were the origin of ECM secretion. In turn, EC-specific
Sox9
inactivation in mice protected from heart failure following TAC or HFD + L-NAME, resp., as indicated by preserved cardiac function, reduced cardiac hypertrophy and fibrosis. In-depth analysis of both bulk and SC RNAseq of isolated cardiac ECs revealed that Sox9 regulates mesenchymal, inflammatory and cell-to-cell communication-associated gene ontologies. Interestingly,
Sox9
expression was limited to a specific EC cluster following TAC, in which
Sox9
and ECM genes were co-expressed. Endothelial
Sox9
inactivation, markedly reduced ECM gene expression in ECs from this cluster, including
Ccn2
, which encodes for CTGF. Both ATAC and CUT&TAGseq of transgenic mice suggested direct induction of
Ccn2
by Sox9 in cardiac ECs. Interestingly, fibroblasts (FBs) co-cultivated with SOX9-overexpressing HUVECs displayed increased levels of mesenchymal gene expression and enhanced migration. This effect was mitigated by knock-down of
CCN2
, suggesting that SOX9 triggers CTGF-mediated signaling from ECs leading to paracrine pro-fibrotic activation of FBs.
In conclusion,
ECs are critical drivers of fibrotic cardiac remodeling by upregulating Sox9, which triggers EndoMA in ECs, leading to ECM secretion from ECs and paracrine FB activation.
Abstract GS.11: Exercise Therapy During Doxorubicin Treatment Provides Sex-specific Cardioprotection
Authors: Tang Lichao , De Melo Amaral Paloma , Efimov Igor , George Sharon Ann
Published: October 2023

Moderate-intensity exercise is routinely prescribed to patients with cardiovascular diseases to improve cardiac function. It has also been recommended in patients undergoing anti-cancer chemotherapy to attenuate cardiotoxic effects. We recently identified that moderate endurance exercise in healthy mice causes the remodeling of cardiac physiology in a sex-specific manner. Therefore, we hypothesized that exercise therapy applied during doxorubicin chemotherapy would produce sex-specific cardioprotection. Male and female mice were divided into three groups, 1) sedentary control, 2) DOX - doxorubicin treated at 5 mg/kg/week for 6 weeks, and 3) EXDOX - 2 weeks of forced treadmill running for 2 weeks before DOX + exercise for an additional 6 weeks. Mouse health was tracked daily. Total body weight, echocardiography, and electrocardiography were assessed every 2 weeks. At the end of the protocol, triple parametric optical mapping was performed to determine the remodeling of cardiac metabolism-excitation-contraction coupling (MECC). DOX treatment increased morbidity and reduced total body weight in both males and females. DOX-induced action potential duration (APD) prolongation in both sexes and, additionally, in males alone, increased anisotropic ratio of conduction (index for arrhythmogenicity) and rise time of calcium transients (CaRT). In EXDOX mice, the onset of morbidity was delayed in both males and females compared to DOX mice. Total body weight in male EXDOX mice, but not females, was further reduced compared to DOX treatment alone. Echocardiography revealed increased LV wall thickness in male and female EXDOX mice vs DOX, while stroke volume was reduced in females alone. Next, DOX-induced APD prolongation was prevented in EXDOX females but not males. Furthermore, DOX-induced increase in CaRT was further increased in EXDOX males, in addition to increased calcium transient duration and reduced calcium decay constant. Taken together, these data suggest significant sex dimorphism in cardiac adaptation to DOX treatment and exercise therapy during DOX treatment. Female mice undergoing exercise therapy during DOX treatment demonstrated greater cardioprotection and attenuation of MECC remodeling.
Abstract P1013: Identification Of Genetic Links Between Cardiovascular Disease And Insomnia In
Drosophila
Authors: Abou Daya Farah , Mandigo Torrey , Maher Matthew , Ober Lily , Patel Dev , Walker James , Saxena Richa , Melkani Girish
Published: October 2023

Cardiovascular disease (CVD) is the leading cause of death worldwide. CVD is associated with insomnia such that insomnia symptoms double the risk of incident CVD. However, how they are related is unknown. Two recent genome-wide association studies (GWAS) identified genetic loci significantly associated with insomnia, including one locus with five nearby genes that are associated with CVD in an independent GWAS. Therefore, we hypothesized that genetic predisposition to insomnia contributes to the development of CVD. To test this and identify causal genes at this locus, we used Drosophila melanogaster, which are well-established model systems for sleep and cardiac studies. To assess the role of these genes on cardiac physiology, we used the cardiac-specific Hand-Gal4 driver to perform genetic knockdown (KD). One-week-old Drosophila progeny were then used for semi-intact microscopic heart preparation followed by high-speed videography to assess cardiac physiology. Similarly, to assess their role in sleep, we used the neuronal-specific Elav-Gal4 driver to perform KD of these genes. Sleep and locomotor activity of one-week-old flies were monitored using the Drosophila Activity Monitoring System. We found that neuronal and cardiac-specific RNAi KD of four genes conserved in Drosophila: Lsn, ATPSynC, Bruce, and Imp, contributes to compromised sleep and cardiac performance, respectively. Cardiac-specific KD of Lsn led to significant cardiac dilation and reduced fractional shortening, a measure of cardiac performance. KD of ATPSynC led to significantly reduced fractional shortening without dilations. Furthermore, Lsn and ATPSynC KD hearts had disrupted actin-containing myofibrillar organization. Suppression of Lsn increased Pericardin deposition, indicative of fibrosis. Also, the cardiac suppression of ATPSynC and Lsn significantly shortened lifespan. Neuronal-specific KD of three genes significantly disrupted sleep. Furthermore, KD of ATPSynC and Lsn in the heart disrupted sleep in a non-cell-autonomous manner. This work provides novel insights into genetic mechanisms linking CVD and insomnia, highlighting the potential importance of these four genes in the development of both diseases.
Abstract P1017: Post-GWAS Functional Analysis Of CBLN2 Locus Reveals The Role Of CUX1 In The Inflammatory Pathogenesis Of Pulmonary Arterial Hypertension
Authors: Sun Wei , Jiang Danli , Wu Ting , Li Gang , Chan Stephen Y
Published: October 2023

Background:
Pulmonary arterial hypertension (PAH) is a deadly pulmonary vascular disease highly relevant to genetic susceptibility and acquired pathogenic triggers, especially inflammation. Chr18q22.3 near cerebellin 2 (CBLN2) gene is the first genetic locus identified by Genome-wide Association Study (GWAS) that is significantly associated with PAH risk in population of European ancestry. This locus includes a group of closely linked non-coding intergenic single nucleotide polymorphisms (SNPs) downstream of CBLN2. Although CBLN2 is upregulated in PAH and causes pulmonary vascular dysfunction, it remains unknown how these non-coding SNPs remotely affect CBLN2 and in turn alter the genetic susceptibility of PAH.

Methods and Results:
We applied Hi-C analysis, combined with electrophoretic mobility shift assay, to identify rs11151770 as a functional SNP (fSNP) characterized with allele-imbalanced nuclear protein binding and chromatin interaction with CBLN2 promoter, among 19 haplotype SNPs in a linkage disequilibrium (LD r
2
>0.8) in CBLN2 locus. As an intergenic non-coding SNP, rs11151770 is located 110 kbp downstream of CBLN2 promoter. Proteomic analysis revealed nuclear transcription factor CUX1 binds specifically to the risk allele A of rs11151770 but not to the non-risk allele C. Acting as the activator for the expression of target gene CBLN2, CUX1 knockdown prevented, but CUX1 overexpression induced, the pathophenotypes of human pulmonary arterial endothelial cells (PAECs) associated with PAH. CUX1-CBLN2 was upregulated by acquired inflammatory PAH trigger IL-1β and was increased in lung tissues extracted from PAH patients. SNP genotyping in PAH patients and non-PAH controls showed a 2.2-fold risk allele A enrichment only in patients with PAH associated with connective tissue disorders, supporting a pathogenic mechanism of an inflammation-sensitive pathway (CUX1-CBLN2) combined with a PAH susceptible genotype (risk allele A of rs11151770) for the manifestation of clinical PAH.

Conclusion:
Our post-GWAS functional genomics analysis revealed a CUX1-mediated inflammatory pathway and provided a mechanistic explanation for the GWAS-identified association between the genetic variants in CBLN2 locus and PAH pathogenesis.
Abstract P1024: Cardiomyocyte Mechanics In Animal Models Of Heart Failure With Preserved Ejection Fraction
Authors: Jani Vivek , Fenwick Axel , Mishra Sumita , Sharp Thomas E , Doiron Jake , LaPenna Kyle B , Goodchild Traci T , jiang nan , Gillette Thomas G , Hill Joseph A , Lefer David J , Kass David A
Published: October 2023

Heart failure with preserved ejection fraction (HFpEF) has few effective therapies yet exacts substantial mortality. Its multi-system nature has made animal modeling difficult, but recent efforts combining diet-induced obesity and hemodynamic stress are popular: mouse - L-NAME/high-fat diet (HFD) and ovariectomized (OVX) females with HFD-induced obesity/cardiac pressure overload (PO) (HFD+OVX+PO), ZSF1 rat, and obese/hypertensive Göttingen minipigs. We reported human HFpEF myocyte defects including reduced Ca
2+
-activated maximum tension (T
max
) negatively correlate with body mass index, reduced Hill coefficient (n
H,
e.g., cooperativity) and Ca
2+
at 50% T
max
(EC
50
), and higher resting tension. Here we tested whether such deficits are found in HFpEF animal models. The L-NAME/HFD mouse had no differences in Ca
2+
-activated (p=0.20) or resting (p=0.75) tension. In obese ZSF-1 rats, while T
max
(p=0.23) and resting tension (p=0.39) were not different, n
H
was reduced (2±1 vs. 4±2 p=0.01). In HFD+OVX+PO mice, T
max
(15±2 vs. 20±2 mN/mm
2
, p=0.006) and EC
50
(1.9±0.6 vs. 2.4±0.7 μM, p=0.008) were reduced, but resting tension (p=0.70) was not different. In the Göttingen minipig HFpEF model, EC
50
(p=0.93) and n
H
(p=0.35) were not different, whereas T
max
was reduced (17±3 vs. 27±3 mN/mm
2
, p=2x10
-6
) and tension less at physiologic Ca
2+
. Resting tension was lower (not greater) in this model (p=0.001). The HFD+OVX+PO and minipig HFpEF models recapitulate depressed Ca
2+
-activated tension as in obese human HFpEF, whereas none manifested increased resting tension (
Table 1
). Studies investigating HFpEF sarcomere dysfunction should consider these findings in model selection.
Abstract P1025: Hsp70 Co-chaperones Regulate Sarcomeric Proteostasis In Hypertrophic Cardiomyopathy
Authors: Wagner Marcus J , Branscom Graham A , Alcaro Liana T , Yob Jaime M , Day Sharlene M
Published: October 2023

Significance:
Recent Genome Wide Association Studies (GWAS) identified non-sarcomeric variants in hypertrophic cardiomyopathy (HCM) with three of the top 10 variant loci in or near genes that encode for co-chaperones of HSP70: BAG3, DNAJC18 and HSPB7. These variants are also present in dilated cardiomyopathy (DCM), but have an opposing effect on cardiac function, leading us to hypothesize that GWAS variants in HSP70 co-chaperones can affect the proteostasis of contractile proteins in the HCM heart. As a first step, we determined the effects each HSP70 co-chaperone knockdown (KD) had on sarcomeric and z-disc protein expression and the poly-ubiquitinated state of cardiomyocyte-specific proteins.

Methods and Results:
Transduction of human iPSC-CMs (iPSC-CMs) with adenoviral vectors driving shRNA expression against BAG3, DNAJC18, HSPB7, or scramble (control) yielded transduction efficiencies
>
75% and >80% reduction in target transcript 4 days post-transduction. BAG3 KD increased poly-ubiquitinated proteins by 38+/-9% (p<0.001) and reduced steady state expression of sarcomeric and z-disc proteins, including: MyBP-C (p<0.0001), actin (p<0.0001), cardiac troponin I (p<0.05), tropomyosin (TPM1) (p<0.01), myosin light chain 3 (MYL3) (p<0.0001), filamin C (p<0.05), and alpha-actinin-2 (p<0.0001). DNAJC18 KD increased poly-ubiquitinated protein by 66+/-17% (p<0.01) but did not impact sarcomere protein expression. Conversely, HSPB7 KD reduced poly-ubiquitinated proteins by 21+/-7% (p<0.01) and increased protein expression of MyBP-C (p<0.01), TPM1 (p<0.05), and myosin light chain 2 (p<0.05). DNAJC18 KD increased BAG3 and HSPB7 protein expression levels by ~2-fold (p<0.001) without increasing transcript levels, suggesting BAG3 and HSPB7 may be protein clients of DNAJC18.

Conclusions:
HSP70 co-chaperones that have an established genetic association with human HCM play an important role in cardiomyocyte proteostasis. Multiple sarcomeric and z-disc proteins appear to be clients of BAG3, while BAG3 and HSPB7 themselves may be clients of DNAJC18. Targeting this axis could be therapeutic for patients with HCM that possess genetic variants in sarcomeric proteins.
Abstract P1028: Elucidating Differential Mechanisms That Underlie Phenotypic Variability In Two Independent Dcm Models
Authors: Vasquez Catherine , Lynn Melissa L , Tardiff Jil C
Published: October 2023

Mutations in components of the cardiac thin filament cause disease via complex, multi-faceted myocellular mechanisms, ultimately presenting as heterogeneous, symptomatic cardiomyopathies. The heterogeneity of both the myocellular mechanism(s) involved and the clinical outcome(s) represents a challenge in ongoing efforts to link genotype to phenotype. Two DCM-associated thin filament mutations (D230N-Tm and R173W-cTnT) with similar clinical phenotypes, were selected explore the molecular basis of disease trajectory. In patient populations, D230N-Tm exhibits an earlier time of onset compared to R173W-cTnT, with children (<1yr) presenting with an often-fatal DCM. We hypothesize that the difference in time of onset can be explained by distinct myocellular mechanisms. We utilized transgenic mice expressing D230N-Tm and R173W-cTnT and defined the progression of pathogenic remodeling across 3 independent timepoints via 2D-echo. At 2 months the %FS of D230N-Tm was significantly reduced (21.79 ± 1.19) compared to NTg littermates (33.45±1.45). R173W-cTnT, however, exhibited no reduction in %FS until 4 months (22.53±0.78), thus phenocopying the patient population. Using these timepoints to guide our myocellular mechanics, contractility measurements were then utilized to assess the myocellular mechanics associated with the mutations. At 2 months D230N-Tm exhibited a decrease in the rate of contraction (4.53±0.24μm/sec) and rate of relaxation (2.88±0.23) compared to NTg. R173W-cTnT did not diverge from NTg until 4 months, exhibiting a later onset of impaired contractility. By 4 months, both mutations displayed the opposite, an increase in rate of contraction and relaxation (rate of contraction, D230N-Tm 4.49±0.18 and R173W-cTnT 5.83±0.27; and rate of relaxation, D230N-Tm 3.14±0.19 and R173W-cTnT 3.70±0.24). Our results suggest that the accelerated time course for the development of D230N-Tm – linked DCM is caused by differential myocellular mechanism(s) that induce the early reduction in contractility and relaxation observed. Ongoing studies aim to further identify possible myocellular mechanism(s) by which these mutations exhibit different myocellular mechanics.
Abstract P1033: Derivation Of Large Biorepository Of Human Ipsc Lines For Open Access Sharing Among Academic Investigators
Authors: Yan Christopher D , Lai Celine J , Shenoy Sushma P , Lee Chelsea , Guevara Julio V , Zhuge Yan , Pang Paul D , Wu Joseph C
Published: October 2023

Background:
Human induced pluripotent stem cells (iPSCs) and their ability to differentiate into numerous cell types have revolutionized biomedical and translational research in many fields such as cardiovascular disease, development, aging, regeneration, cancer, personalized medicine, and many rare diseases. The need for obtaining iPSC lines with the associated donors’ clinical data and from patients of diverse genetic backgrounds has been greater than ever. Greenstone Biosciences Inc. (GSB) has built a curated biorepository to address this demand as an asset for the scientific community utilizing iPSCs.

Methods:
The GSB biobank curates clinical data, isolates peripheral blood mononuclear cells (PBMCs), reprograms and banks patient-specific iPSC lines, and performs quality assurance. Reprogramming of PBMCs to iPSCs is carried out using non-integrating Sendai virus encoding for Oct4, Sox2, Klf4, and c-Myc. We then rigorously validate all iPSCs for pluripotency using immunofluorescence of stemness markers, RT-PCR, and SNP-based virtual karyotyping along with quality control methods established as a standard protocol.

Results:
We have a growing repository of >1,000 iPSC lines with various disease backgrounds across demographics such as age, sex, race, and ethnicity. The diseases with genetic mutations include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), long QT syndrome (LQTS), arrhythmogenic right ventricular dysplasia / cardiomyopathy (ARVD/C), Brugada syndrome (BrS), Duchenne’s muscular dystrophy (DMD), Spinal muscular atrophy (SMA), and many other rare diseases and syndromes. We have also established a simple material transfer agreement (MTA) for open access by academic investigators.

Conclusions:
We introduce the Greenstone Biosciences iPSC Biobank, a biorepository containing >1,000 human iPSC lines with de-identified patient clinical information and genetic backgrounds. This invaluable resource will help support iPSC research among academic investigators.
Abstract P1057: Outcomes Of Impella Versus ECMO In Multivessel Percutaneous Coronary Intervention
Authors: Khalid Yaser , Dasu Kirti , Amin Hiral , Gabriel Nielsen , Shetty Sanath , Dasu Neethi , Jha Debesh , Klecha Tyler
Published: October 2023

Background:
Impella is a mechanical circulatory support device indicated in severe cardiogenic shock and high-risk percutaneous coronary intervention. ECMO is also used during the percutaneous coronary intervention (PCI) in an acute coronary syndrome that is complicated by refractory cardiogenic shock. There have been few studies examining short-term mechanical circulatory support devices for patients that undergo PCI, so the choice between Impella and ECMO is still a matter of debate. We investigated the in-hospital outcomes of Impella and ECMO in patients who underwent multi-vessel PCI (MVPCI).

Methods:
In a retrospective cohort study of National Inpatient Sample 2017 data, we used ICD-10 codes to identify patients who underwent MVPCI and received Impella or ECMO support. Multivariate logistic regression was used to adjust for patient demographics, hospital demographics, and relevant comorbidities. Primary outcomes (inpatient mortality, length of stay (LOS), and total hospital charges. Secondary outcomes, such as respiratory failure (RF), gastrointestinal hemorrhage, cerebrovascular accident (CVA)/transient ischemic attack (TIA), intracerebral hemorrhage (ICH), acute kidney injury (AKI), ventricular tachycardia (VT), and atrial fibrillation, were also analyzed.

Results:
Among 64,489 patients who had MVPCI, 102 (%) received ECMO support, and 927 (%) received Impella support. Both Impella and ECMO use was associated with high in-hospital mortality (Impella 21.8% vs ECMO (49.5%), high hospital LOS (ECMO 19.9 days vs Impella 9.28 days), and total hospital charges (Impella $3,200,000 vs ECMO $7,500,00). Mortality (OR 4.27, p<0.0001), hospital LOS (OR 5.9, p<0.0001), and total hospital charges (OR 7.52, p<0.0001) for patients undergoing MVPCI. There was no statistically significant difference in complications such as RF, gastrointestinal hemorrhage, CVA/TIA, ICH, AKI, VT, and atrial fibrillation. Hispanic patients had higher mortality with ECMO (OR 2.28, p <0.024) than all other ethnicities and races.

Conclusions:
Both patients with Impella support and ECMO support had significant inpatient mortality. Our analysis suggests that Impella patients had better outcomes than patients on ECMO for MVPCI.
Abstract P1066: Engineering Of Ferritin Overexpressing Human Induced Pluripotent Stem Cells For In Vivo Tracking Of Grafts Using Magnetic Resonance Imaging
Authors: Son Yura , Zhu Wuqiang
Published: October 2023

Introduction:
Transplantation of human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) emerges as a promising approach for the treatment of heart diseases. However, it is an unmet challenge to precisely localize the hiPSC-CMs upon transplantation and quantify the graft size in the live large animals. To address this issue, we established a new approach to track grafted CMs in large animals using magnetic resonance imaging detectable reporter molecules, which allows the assessment of graft size in live animals at different time points.

Methods:
We employed a CRISPR/Cas9 system as a virus-free approach to overexpress ferritin in hiPSCs.

Results:
hiPSC cells were transfected with ferritin heavy chain CRISPR activation plasmid for 24 hours. Before antibiotic selection, we established a kill curve to determine the appropriate antibiotic concentration. Based on the kill curve, we conducted antibiotic selection using puromycin for 7 days and isolated transfected colonies for further culture. Next, we treated various concentrations of iron chloride (0-100 μM) to the FHC transfected hiPSC for 4 hours to examine a proper concentration of iron loading for MRI detection. There was no significant difference in cell viability between 0 and 25 μM of iron chloride. In addition, we confirmed transfection efficiency with higher ferritin expression in transfected cells compared to controls (non-transfected wild-type hiPSC). Following, we plan to differentiate ferritin overexpressing hiPSC into cardiomyocytes, inject the cells into porcine hearts, and conduct magnetic resonance imaging to detect the engrafted transgenic cells in live animals.

Conclusion:
The ferritin-expressing hiPSC line has the potential to provide a reliable measurement of the graft size in large animals.
Abstract P1080: Periostin Is Induced During Cell-based Repair In The Sheep Model Of Myocardial Infarction
Authors: Vega Richard , Hughes Lorelei , Hasaniya Nahidh , Bailey Leonard , Kearns-Jonker Mary K
Published: October 2023

Cardiac disease is the leading cause of death worldwide. Stem cell-based therapy may improve patient outcomes following a myocardial infarction. Our laboratory is studying the mechanistic basis by which neonatal Islet-1 positive cardiovascular progenitor cells (CPC) facilitate post infarct repair in the sheep model of myocardial infarction. Here, we addressed the hypothesis that periostin (POSTN) is induced in the cardiovascular repair zone following neonatal Islet-1+ cell-based repair. POSTN promotes cardiomyocyte proliferation, recruitment of M2 macrophages, epithelial-mesenchymal transition (EMT), migration, and angiogenesis. We examined human neonatal Islet-1+ cardiovascular progenitor cell clones isolated from several patients to initially determine whether or not POSTN was expressed in these cells. All clones expressed POSTN as shown by RT-PCR. The size of the amplified transcripts was confirmed by gel electrophoresis and POSTN expression in these clones was further confirmed by RNA sequencing. We then examined POSTN expression in the cardiovascular repair zone following myocardial infarction and cell transplantation in sheep. Sheep neonatal Islet-1+ CPC clones were used in this model of allogeneic cell-based repair. A myocardial infarction was induced in three Western sheep by left anterior descending coronary artery ligation. Ten million neonatal sheep Islet1+ cardiac progenitor cell clones isolated from 9 day old sheep were injected into the area surrounding the infarct site 3-4 weeks post infarction. RNA was isolated from the cardiovascular repair zone two months after cell injection. The non-infarcted region of the left ventricle and the left ventricle of normal, non-infarcted sheep were used as controls. POSTN transcripts were significantly elevated in the cell-treated cardiovascular repair zone of all three sheep, as identified by qRT-PCR relative to controls. Periostin levels were elevated between 6 and 255 fold in the repair zone relative to the non-infarcted region of the heart. Our data shows that POSTN is expressed in human Islet-1+ neonatal cardiovascular progenitor cells and that POSTN levels are induced during cell-based cardiovascular repair in the sheep model of myocardial infarction.
Abstract P1087: Cd36-retinoic Acid-PPARδ Signaling Axis Modulates Cardiomyocytes’ Ability To Proliferate In Newly Born Neonatal Mice
Authors: Salama Abou Bakr M , Abouleisa Riham , Ou Qinghui , Miller Jessica , Dwenger Marc , Gebreil Ahmad , abdelhafez Hania , Arai Momo , Abdulwali Sameeha , Almasry yazan , Hill Bradford G , Mohamed Tamer
Published: October 2023

Background:
A pertinent question is why only a subset of fetal/neonatal cardiomyocytes (CMs) spontaneously proliferate. Using single-cell RNAseq, we previously showed a specific population of CMs that is more responsive to cell cycle induction (Abouleisa et al., Circulation, 2022). This population is characterized by expression of the transmembrane scavenger receptor, CD36 (fatty acid (FA) internalization receptor). Here, we aim to investigate the mechanism by which CD36 modulates CMs decision to enter into the cell cycle in neonatal P1 CMs.

Methods and Results:
Our single-cell RNAseq from primary CMs isolated from P1 hearts demonstrated that CD36 is specifically expressed in the spontaneously proliferating CMs and the CMs that respond to the exogenous cell cycle stimulation. CMs isolated from P1 CD36 knockout mice (CD36
KO
) showed 50% less proliferation capacity compared to WT P1 CMs. Interestingly, CD36
KO
mice are born with smaller hearts which contain fewer CMs compared with their WT littermates. Furthermore, compared with WT littermates, which completely regenerate the heart apex following apical resection at P1, CD36
KO
showed minimal regenerative capacity. Surprisingly, CD36
KO
CMs didn’t show significant disruption in FA oxidation or glycolysis; however, bulk RNAseq data from the P1 CD36
KO
hearts showed significant downregulation in the expression of retinoic acid (RA)-dependent signaling genes (e.g., PPARδ and RXRa) and a significant increase in PPARα expression. Confirming a causal role of CD36 in CM proliferation, we found that Cd36 is crucial in regulating the entry and interactions of RA or retinoid precursors in CMs. The CD36
+
FACS-sorted CMs express significantly higher levels of PPARδ, RXRa, and FABP5 and lower levels of PPARα, which are crucial in regulating the response to cell proliferation. Furthermore, small molecule activation of PPARδ doubled cell cycle entry in P1 CMs and completely restored proliferation in CD36
KO
CMs. These data suggest that CD36 is essential to prime CMs proliferation during development by regulating RA/PPARδ/RXR transcriptional signaling.

Conclusions:
These findings suggest that the CD36-RA-PPARδ signaling axis modulates the CMs’ ability of P1 to spontaneously enter the cell cycle.
Abstract P1092: Endothelial Cell Aging Can Be Reversed By A Pharmacological, Partial Reprogramming Strategy
Authors: Kalies Katrin , Knoepp Kai , Wurmbrand Leonie , Dutzmann Jochen , Sedding Daniel
Published: October 2023

Senescent endothelial cells (EC) are key players in the pathophysiology of cardiovascular diseases and characterized by a reduced angiogenic and regenerative potential. Therefore, reversing EC senescence represents a promising therapeutic strategy to restore vascular integrity and increase health and lifespan. Here, we show a reversal of EC senescence following the application of a pharmacological, non-genetic, partial reprogramming strategy to induce a timely restricted induction of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM).

Methods to characterize the effects of pharmacological reprogramming included the quantification of gene expression as well as functional analysis of EC
in vitro
. In addition, the regenerative capacity of EC was evaluated in a hind-limb ischemia model
in vivo
in old C57BL/6J mice.

We demonstrate that the application of a pharmacological cocktail of FDA approved drugs to senescent EC resulted in a robust but timely restricted induction of OSKM (p<0.001). Additionally, siRNA-knockdown prior to the treatment confirmed the correlation between OSKM upregulation and pharmacological treatment. Induction of OSKM was associated with a significantly reduced expression of senescence markers as p16ink4a and p14arf (p<0.001). Expression of cytokines as TNFa or Il-6 was significantly reduced compared to untreated senescent cells (p<0.05). Further the telomere length was stabilized (p<0.05). We also observed enhanced functional properties of senescent EC, such as proliferation, migration, sprouting and tube formation (p<0.05). Long term cultivation of treated EC indicated that expression of p16ink4a and p14arf remained low (p<0.05), and the migratory capacity was preserved. Non-senescent EC were not influenced by the pharmacological treatment.
In vivo
, a significantly improved blood flow was observed after hind limb ischemia in 22 months old C57BL/6J mice after 7 and 14 days (p<0.001). Capillary density was significantly enhanced (p<0.05).

In conclusion, we demonstrate that a short induction of OSKM via a pharmacological approach holds the potential to reverse senescence in EC
in vitro
and thus to enhance endothelial regenerative capacity
in vivo
. This might present a promising strategy for ischemic diseases.
Abstract P1095: Studying Changes In Mitochondrial Dynamics And Energetics During Cardiac Reprogramming
Authors: Spurlock Brian , Xie Yifang , Salmenov Rustem , Liu Jiandong , Qian Li , Colon Marazzano
Published: October 2023

In mammals, cardiac ischemic injury leads to loss of muscle filled in with less functional scar tissue. Direct reprogramming of the cardiac fibroblasts populating a scar to functional cardiomyocytes represents a promising potential treatment for these patients. This reprogramming involves a metabolic shift from the more glycolytic CF state to mitochondrial oxidative phosphorylation in the induced CMs, including increased mitochondrial biogenesis. Various reprogramming strategies make use of agents, which in addition to canonical roles, promote mitochondrial energetics and a dynamic shift towards mitochondrial fusion. We decided to investigate whether these mitochondrial structural and functional shifts might represent a mechanism of reprogramming. We characterized various structural and energetic properties of mitochondria in neonatal CFs, CMs, and at multiple time points during reprogramming. Using the baseline CF and CM mitochondrial data, we built a model to follow cells in pseudotime during reprogramming, and identified a time course for mitochondrial changes. We also investigated whether various mitochondrial perturbations designed to promote these changes might increase reprogramming efficiency. Promotion of mitochondrial fusion activity and biogenesis increased the proportion of reprogrammed cells. Conversely, promotion of fission and inhibitors for electron transport system enzymes inhibited reprogramming.
Abstract P1096: Computational Motion Artifact Removal Acts As Virtual Blebbistatin In Ipsc-derived Cardiomyocytes And Micro-tissues
Authors: Woodhams Louis , Guo Jingxuan , Huebsch Nathaniel , Genin Guy
Published: October 2023

Introduction:
Drugs like blebbistatin that paralyze tissues are critical for accurate optocardiography but directly affect sarcomeres, making it challenging to study structure-function relationships. To overcome this, we applied Direct Deformation Estimation (DDE), a robust strain estimation tool, to enable computational motion removal from videos of beating monolayers and engineered heart tissues formed from human Induced Pluripotent Stem Cell derived cardiomyocytes (hiPSC-CM).

Methods:
hiPSC-CM and miniaturized engineered heart muscles were stained with the far-red voltage sensitive dye, BeRST-1. Videos of overall motion (bright-field), Ca
2+
(endogenous GCamP6f reporter) and voltage were captured at 100 Hz. Temporal median filtering and Contrast-Limited Adaptive Histogram Equalization were applied as pre-processing steps to improve convergence. The inverse of conformal mappings estimated from DDE were used to “unwarp” videos to artificially reduce motion. Removal of motion was assessed in bright-field videos by performing optical flow analysis. Improvements in optical mapping accuracy were estimated by calculating the spatial distribution of APD
80
(voltage) and time to 75% decay from peak (τ
75
; Ca
2+
).

Results:
Virtual blebbistatin removed >90% of motion from monolayers and engineered tissues without affecting sarcomere architecture (sarcomeric α-Actinin staining), in contrast to chemical blebbistatin. Virtual blebbistatin further reduced spatial dispersion in both APD
80
and τ
75,
leading to overall distributions that were less skewed by artificially low repolarization times. Interestingly, when directly assessing Ca
2+
-contraction coupling in plots of motion-corrected Ca
2+
vs. contraction magnitude, we observed that engineered tissues had more uniform and consistent coupling compared to monolayers.

Conclusions:
Superior uniformity in calcium-contraction coupling of engineered tissues as opposed to monolayers is consistent with observations that engineered tissues are more likely to recapitulate genotype-phenotype relationships and drug responsiveness. Virtual blebbistatin is a promising tool for non-invasive analysis of Ca
2+
-contraction coupling and robust optocardiography.