bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract We036: Recapitulating Cardiac Microenvironment: Elucidating the Role of ECM Components and Architecture on Cardiac Differentiation
Authors: Ali Kiran , Gluck Jessica
Published: October 2024

The discovery of iPSCs was revolutionary for regenerative medicine, especially as a source to replace a cell population with limited regeneration capability, like cardiomyocytes (CMs). However, the exact mechanism(s) governing how the microenvironment influences cardiac differentiation at a cellular scale is still widely unclear. To bridge this gap, we created an in vitro fibrous model from a decellularized porcine cardiac extracellular matrix. The aim is to assess the impact of the microenvironment, specifically the effects of protein composition and micro-architecture, on the cardiac differentiation of iPSCs. To study the role of protein composition, atrial and ventricular regions of the porcine heart are manually identified and separated as their protein composition is observed to be different. To test this, the material was subjected to proteomics analysis with LS-Mass Spectrometry where about 1000 proteins were identified in the ventricular region and only 300 proteins in atria. The decellularized tissue was tested for complete removal of nuclei by staining and further validated with DNA quantification. The dECM was blended with synthetic polymer (polycaprolactone) for electrospinning. The average collected fiber diameter was 14.8 ± 3.9 μm. The fibrous scaffolds were seeded with iPSCs and differentiated towards cardiac lineage. dECM samples show comparable signs of biocompatibility as laminin-521 coated TCPS, forming clusters throughout the differentiation process indicating that the scaffolds encourage cardiac differentiation. Further analysis is being conducted to compare the cellular expressions of differentiation markers including mesoderm formation, cardiac specification, and cardiomyocyte structural and functional markers. The effects of the ECM composition will be checked with myosin light and heavy chain expressions, as their expressions vary in the atrial and ventricular regions of the heart.
Abstract We040: Molecular Characterization of Human Highly Proliferative Cells Isolated from Coronary Artery Bypass Graft Surgery Patients Identifies IGFBP3, SOST, and ISLR as Downstream Targets of BMP9/ALK1 Signaling
Authors: Moore Michayla , Ryzhov Sergey , Sawyer Douglas , Vary Calvin
Published: October 2024

Stem/progenitor cells are being investigated as a potential cell source to reverse cardiac remodeling and restore cardiac function, but the molecular mechanisms required for improved repair are not well understood. Circulating BMP9 is negatively associated with heart disease risk factors. BMP9/ALK1 signaling is a critical pathway involved in several mechanisms of vascular repair such as angiogenesis and inflammation. We recently identified ALK1 expression in human highly proliferative cells (hHiPCs) with progenitor capabilities isolated from patients undergoing coronary artery bypass graft (CABG) surgery as measured by flow cytometry. We have determined that circulating BMP9 in patients is negatively associated with number of hHiPC (p<0.001) and positively associated with endothelial cell (EC) number (p=0.008) suggesting that BMP9 may play a role in hHiPC differentiation into EC. Further, we found upregulation of novel BMP9 regulated proteins, IGFBP3, SOST, and ISLR, in hHiPC upon BMP9 treatment using LC-MS/MS analysis of the secretome (p<0.01, Fold-change (FC)>2.0) and RT-qPCR (p<0.001, FC>2.0). Conditioned media collected from BMP9 treated hHiPCs or ECs improved tube formation in the tube formation assay. IGFBP3, SOST, and ISLR upregulation by BMP9 is inhibited upon knockdown and pharmacological inhibition of ALK1 as measured by proteomics (p<0.01) and RT-qPCR analysis (p<0.05). ISLR is a novel marker of mesenchymal stem cells found to have an important role in maintaining “stemness” and colony formation. hHiPCs treated with novel BMP9 target, ISLR, were analyzed using LC-MS/MS and STRING. We found ISLR treatment enriched for differential regulation of cell adhesion, cell migration, and angiogenesis proteins such as CXCL12, EMILIN1, and ANXA5 (p<0.05). Together, these data lay the foundation for investigating BMP9/ALK1 autocrine signaling in potential regulation of progenitor cell maintenance and angiogenesis in heart disease.
Abstract We047: Enhanced Vascularization in hPSC-derived Cardiac Organoids by Dynamic Culture
Authors: Huerta Gomez Angello , Kang Soo-Yeon , El-Mokahal Marcel , Lee Moo-Yeal , Yang Huaxiao
Published: October 2024

Introduction:
Human pluripotent stem cells (hPSC)-derived cardiac organoids (COs) have gained increasing interest in biotechnology due to their characteristics like diverse cardiovascular cell types, 3D complex structure, and function that recapitulates the human heart in early development. However, current methods for vascularizing COs are not systematic and robust.

Hypothesis:
We hypothesize that culturing hPSC-derived COs dynamically will further enhance vascularization in COs.

Materials and Methods:
Two conditions were created to compare vascularization in COs: 1) dynamic culturing (Dyn) and 2) static culturing (St). Firstly, hPSCs were cultured for two days in ultra-low attachment plates to develop spheroids. The spheroids were then transferred to pillar plates followed by direct cardiac differentiation the next day. Live-cell imaging was used to monitor the process of vascularization for up to 16 days and the corresponding fluorescence intensity of each cell type from its reporter expression was analyzed. The cardiac function was measured by video-based contractility analysis. Data were presented as mean ± standard error of the mean (SEM). The student’s t-test was used for the statistical analysis.

Results:
Cardiomyocytes (CMs) and endothelial cells (ECs) were imaged on pillars under the two conditions as shown in
Fig. 1A
. Successful cardiac differentiation was present, displaying hPSC-CM contraction on day 12 and onwards. Higher intensity values of hPSC-CMs under Dyn were observed in all four days compared to St as shown in
Figs. 1B&C
. The hPSC-ECs intensity displayed greater intensity values compared to St, demonstrating enhanced vascularization in VCOs due to the active Dyn as shown in
Figs. 1D&E
. The functionality was improved in Dyn as indicated with higher contraction velocity as shown in
Fig. 1F.

Conclusion:
This study demonstrates the effects of incorporating dynamic culture for enhancing cardiogenesis and vasculogenesis in hPSC-derived COs. The enhanced hPSC-VCOs hold promise for studying early cardiovascular development, congenital heart disease modeling, and drug screening and discovery.
Abstract We048: Exon-skipping therapy can restore functional dystrophin attenuating calcium overload for DMD cardiomyopathy with mutations in actin-binding domain 1
Authors: Shiba Naoko , Yang Xiao , Agata Masahiro , Nagamine Kohei , Kadota Shin , Mitsuto Sato , Nakamura Akinori , Shiba Yuji
Published: October 2024

Antisense oligonucleotide (AO)-mediated exon-skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding Dp427m, leading to progressive cardiomyopathy. In-frame deletion of exons 3–9 (Δ3–9) manifesting asymptomatic or very mild clinical phenotype is an ideal goal for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this therapy for DMD cardiomyopathy remains unclear. We compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3–9, frameshifting Δ3–7, and intact DMD. RNA-seq revealed high similarity between Δ3–9 and wild-type hiPSC-CMs. Furthermore, we observed electrophysiological alterations with accelerated CaMKII activation in Δ3–7 hiPSC-CMs compared to Δ3–9 and wild-type hiPSC-CMs, while Δ3–9 Dp427m was stably expressed, maintaining substantial binding to F-actin and retention of desmin. AOs targeting exon 8 efficiently induced exons 8–9 skipping to restore functional Dp427m and electrophysiological parameters in Δ3–7 hiPSC-CMs. Exon-skipping therapy converting the reading frame to Δ3–9 might be promising for DMD cardiomyopathy.
Abstract We052: Angiotensin-converting enzyme (ACE): A new role to regulate β-oxidation in monocytic cells
Authors: Cao Duo-Yao
Published: October 2024

The metabolic failure of macrophages to adequately process lipid, and the deleterious effects of ingested lipid on macrophage behavior, is central to the etiology of atherosclerosis. As angiotensin-converting enzyme (ACE) has major effects on myeloid cell metabolism, we examined the role of macrophage ACE in a mouse model of atherosclerosis. Animals with increased macrophage ACE expression (ACE 10/10 mice) have a marked reduction in atherosclerosis vs. WT mice. Macrophages from both the aorta and peritoneum of ACE 10/10 express increased PPARα and higher levels of effector molecules downstream of PPARα. These macrophages have increased basal and maximal oxygen consumption compared to WT macrophages. When exposed to lipid in vitro, ACE 10/10 macrophages ingest more fatty acid than WT and have increased efferocytosis. ACE 10/10 macrophages metabolize via β oxidation far more fatty acid than WT cells as determined using 13C isotope tracing. In part by increasing PPARα, ACE boosts myeloid metabolic function under pathophysiologic lipid stress, addressing a key myeloid failure in atherosclerosis.
Abstract We061: Cardiomyocytes HIPK2 Regulates Myocardial Inflammation And Heart Function Through Purinergic Signaling
Authors: Sultan Tousif , Umbarkar Prachi , Sethi Rohan , Toro Cora Angelica , Chen Yunxi , He Jin , Dubey Praveen , zhang qinkun , Xie Min , Krishnamurthy Prasanna , Lal Hind
Published: October 2024

Background:
Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that belongs to an evolutionary conserved HIPK family of kinases. HIPK2-mediated regulation of the inflammation axis of signaling is a comparatively new concept. Purinergic signaling is critical to myocardial inflammation and function. However, there is no literature on HIPK2 regulating purinergic signaling.

Methods and Results:
Herein, we identified HIPK2, as a novel regulator of purinergic signaling. Cardiomyocyte-specific HIPK2 knockout (CM-HIPK2-KO) hearts exhibited reduced expression of multiple key players of canonical purinergic signaling, including ectonucleases CD39 and CD73, and led to excessive inflammation and cardiac dysfunction. Multiple in vitro experiments with gain-of-function (adenovirus expressing HIPK2, Ad-HIPK2), and loss-of-function (Ad-sh-RNA-HIPK2) approaches were performed to establish that HIPK2-mediated regulation of purinergic signaling is a conserved mechanism in many cell types of diverse backgrounds. Mechanistically, we identified that the signaling circuit of HIPK2-ERK-CREB exerts its effects on purinergic signaling through transcriptional control on CD39 and CD73. Aberrant activation of purinergic signaling and inflammation was critical to cardiac pathologies because interventions with purinergic signaling inhibitor apyrase or NLRP3 inflammasome inhibitor CY09 largely rescued the detrimental cardiac phenotype of CM-HIPK2 KOs.

Conclusion:
Herein, we identified HIPK2 as a novel regulator of purinergic signaling. Its deletion leads to excessive inflammation and cardiac dysfunction. Therefore, strategies to maintain HIPK2 are critical to sustaining cardiac health.
Abstract We068: TRAF6 Mediates TFEB Ubiquitination to Inhibit Cardiomyocyte Autophagy in Sepsis-Associated Cardiac Dysfunction
Authors: Wang Xiaoping
Published: October 2024

Sepsis-associated cardiac dysfunction (SACD) is the predominant cause of death in patients with severe sepsis. Complex mechanisms have been proposed for SACD, including maladaptive inflammation and autophagy. However, studies in the past have emphasized these different manifestations underlying SACD as discrete processes and largely neglect their inherent connection. Herein using both in vivo and in vitro SACD models, we showed that the cardiomyocyte Toll-like receptor 4 (cTLR4) can serve as a key receptor mediating the interaction between the cardiomyocyte and macrophage under SACD-like conditions. The stress-induced secretome of macrophage caused activation of cTLR4 which intensified the impairment of autophagic flux in cardiomyocytes. The TNF receptor-associated factor 6 (TRAF6) was further identified as the primary downstream target which conferred the regulatory effects of TLR4 signaling on cardiac autophagy. Importantly, we found that cardiac targeted
Traf6
gain-of function exacerbated, while cardiac targeted
Traf6
loss-of function partially rescued, the cardiac dysfunction in a mice model of SACD. Mechanistically, TRAF6 functioned as a ubiquitin E3 ligase to facilitate the degradation of transcription factor EB (TFEB) via ubiquitination and subsequently led to the blockage of autophagic flux. Along the line, dynamics computational simulation combined with experimental validation revealed unique local electrostatic interactions between human TRAF6 and TFEB. Overall, our study provides novel mechanistic supports which link the macrophage-initiated inflammation and cardiomyocyte autophagy, demonstrates a previously undescribed role of cTLR4/TRAF6/TFEB signaling in fine-tuning cardiac autophagy, and highlights TRAF6 as a novel therapeutic gene target to develop autophagy-based therapeutics for SACD.
Abstract We069: Post-infarction Cardiac Function is Improved in Mice with TSC2
-/-
Macrophages
Authors: Keykhaei Mohammad , Koleini Navid , Meddeb Mariam , Tajdini Masih , Liu Kejun , Kass David
Published: October 2024

Introduction:
Macrophages (MF) play major roles in myocardial ischemia/reperfusion (I/R) injury, both beneficial and detrimental. The mechanistic target of rapamycin (mTOR) plays an important role in MF polarization and functionality. It is prominently regulated by tuberous sclerosis complex 2 (TSC2) which constitutively inhibits and regulates mTORC1. Little is known about how such mTORC1 regulation influences the effect of MFs in I/R injured hearts.

Hypothesis:
We tested if gene knockdown of TSC2 to constitutively enhance MF mTORC1 activity by TSC2 knockdown impairs or benefits the I/R heart.

Methods/Results:
MF-specific TSC2 knock-out mice (MF
TSC2-/-
) were generated by crossing flox’d TSC2 mice with those expressing LysM-Cre. MTORC1 was constitutively active in bone marrow-derived MF
TSC2-/-
, reflected by increased S6K1 and 4E-BP1 phosphorylation. MF
TSC2-/-
exposed to liposaccharide had greater proinflammatory responses, e.g. increased TNF-a secretion, supporting an M1-like phenotype. MF
TSC2-/-
also exhibited less M2-like features (reduced Arg1, Mrc1, and Rentla expression). To test the impact of MF
TSC2-/-
on infiltrating myocardial MF subtypes post I/R, MF were isolated from the myocardium and sorted by flow-cytometry based on CD45
+
, CD11b
+
, and CD64
+
and then parsed as ±CCR2 and ±MHC-II. We found MF
TSC2-/-
resulted in an increase in infiltrating CCR2
-
MHC-II
-
phenotypes, which are thought to be anti-inflammatory and to support healing. Consistent with this, we found MF
TSC2-/-
hearts exposed to I/R had higher ejection fractions (74.2±10.3%) vs controls (47.2±14.3%, P=0.002). MF
TSC2-/-
hearts also had smaller LV internal end-systolic diameter (P=0.009). As LysM is also expressed in neutrophils, we also performed this study in mice where neutrophils were first depleted using an anti-Ly6G antibody. Similar cardioprotective effects were observed with MF
TSC2-/-
after I/R: (EF: 56.9±12.3 vs 36.1±9.8, P=0.01). This supports the impact being conferred by MF.

Conclusion:
TSC2-deleted MF with constitutive mTORC1 activation confer cardio-protection to I/R injury. These findings help identify a novel signaling mechanism coupling mTORC1/TSC2 in MF to myocyte recovery, highlight novel avenues to improve post ischemic cardiac repair.
Abstract We071: Identifying Preventive and Therapeutic Effects of SGLT2 Inhibitor on Atrial Fibrillation
Authors: Park Hyewon , Park Bokyeong , Park Hyelim , Park Junbeom
Published: October 2024

Background:
The frequency and prevalence of atrial fibrillation (AF) and heart failure are expected to increase over time, which are expected to result in enormous medical expenses and socioeconomic losses. Previous studies have confirmed that SGLT2 inhibitor (SGLT2i) reduces cardiovascular mortality and hospitalization rates in heart failure patients. Also, follow-up studies have been reported that the use of SGLT2i reduces atrial fibrillation, and its administration is recommended in clinical practice guidelines. In spite of beneficial effects of SGLT2i on atrial fibrillation, the exact mechanism by which SGLT2i reduces atrial fibrillation has not been revealed.

Methods:
We performed experiments by creating an AF cell model using Ang II and an AF animal model using Acetylcholin-Calcium chloride. Fibrosis markers and calcium regulatory proteins were confirmed by Western blot, and the occurrence and burden of AF were evaluated by using a 1F catheter in animal models.

Results:
In our cell studies and animal experiments, the inhibitory effect of SGLT2i administartion on atrial fibrillation was confirmed. First, fibrosis markers tend to reduce in atrial fibrillation models treated with SGLT2i, compared to non-treated atrial fibrillation models. In addition, the phosphorylation of the calcium handling proteins, such as CaMKII, RyR2, and PLB, increased in the AF group, whereas the degree of phosphorylation in SGLT2i treated group decreased to the phosphorylation level in control group.

Conclusion:
In AF-induced cell and animal models, the effect of SGLT2i was shown to be highly effective against atrial fibrillation. In particular, the results indicate that administration of SGLT2i before inducing atrial fibrillation could bring out the preventive and therapeutic effects through amelioration of fibrosis and reduction of calcium handling proteins phosphorylation.
Abstract We082: Contribution of local hematopoiesis to endocardial cushion remodeling during development
Authors: Liu Norika , Nakano Atsushi
Published: October 2024

Introduction:
The cells that comprise the circulatory system not only share their origins but also mutually promote each other’s differentiation during the formation of functional circulatory system. We previously reported a subset of endocardial cells is hemogenic during early embryogenesis. They are enriched in the cushion region, the primordia of the cardiac valves and septa, where active remodeling via endothelial-mesenchymal transition takes place. Hemogenic endocardial cells undergo endocardial-hematopoietic transition (EHT) via Nkx2-5-dependent manner, suggesting that Drosophila tinman-dependent cardio-hematopoietic program is conserved in mammals.

Methods:
In this study, we analyzed the regulatory network of Nkx2-5-dependent endocardial hematopoiesis using a scRNA-seq data from wildtype and Nkx2-5-null embryonic hearts. In vivo genetic analysis were performed to confirm the candidate signal pathways driven under Nkx2-5 transcription.

Results and Conclusion:
Nkx2-5-null hearts were devoid of clusters for hemogenic endocardium and cushion endocardium. Interestingly, scRNA-seq analysis further revealed that genes related to Notch signaling pathway are significantly downregulated in Nkx2-5-null endocardium. A further gene network analysis identified that Dhrs3, an enzyme that attenuates retinoic acid (RA) signal by catalyzing the reduction of all-trans-retinaldehyde to all-trans-retinol, is a signature gene of the hemogenic endocardial cells downstream of Nkx2-5. Although RA signal is known to induce the formation of multipotent progenitor, our
ex vivo
hematopoietic colony forming assay revealed that macrophage formation is strongly inhibited by RA signal. Notch inhibition also suppressed the formation of macrophages. Consistently,
in vivo
forced activation of NICD drastically increased the number of CD41
+
hemogenic endocardial cells as well as macrophages in the cardiac cushion. Strikingly, impaired hematopoiesis and cushion defects in the Nkx2-5-null heart were both rescued by overexpression of NICD, suggesting that Notch signaling promotes endocardial hematopoiesis downstream of Nkx2-5. Taken together, our study demonstrated that the Nkx2-5/RA/Notch signaling axis plays a pivotal role in EHT during early embryogenesis, thereby facilitating local tissue remodeling by inducing macrophage differentiation.

The talk will include the latest findings on the specific role of endocardially-derived tissue macrophages.
Abstract We092: Naltrindole Attenuates Superoxide Release in Polymorphonuclear Leukocytes by a Novel Mechanism devoid of Delta opioid receptor antagonism Related to Reduced Intracellular Calcium Levels
Authors: Le Mai An , Dang Jennifer , Stinson Cameron , Johnson Devani , Clair Logan , Ramsarran James , Boakye Tanoh Desmond , Humayun Annam , Melnik Juliet , Dean Tameka , CHEN Qian , Barsotti Robert , Young Lindon
Published: October 2024

Introduction:
Naltrindole (NTI), known as a delta opioid receptor antagonist, inhibited phorbol 12-myristate 13-acetate (PMA) induced superoxide (SO) release in polymorphonuclear leukocytes (PMNs). PMNs do not express opioid receptors. We propose NTI reduces PMN SO by reducing intracellular calcium (Ca
2+
). To test this hypothesis, we did experiments using KB-R7943 (KB) as a positive control, known to reduce intracellular Ca
2+
via inhibition of the reverse mode Na
+
/Ca
2+
exchanger. Naloxone (NX), a broad-spectrum opioid receptor antagonist, was used as a negative control. We predict that NTI will diminish PMA-induced PMN SO release similar to KB, while NX will have no effect, and that there will be no difference in cell viability compared to vehicle controls.

Methods:
Male Sprague Dawley (SD) rat PMNs (5x10
6
) were incubated for 15 min at 37
o
C in the presence or absence (dH
2
O vehicle control) of NTI (10
-
200 µM), KB (5-20 µM), and NX (100-200 µM). PMN SO release was measured by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after PMA stimulation (100 nM). The cell viability was determined microscopically by 0.2% trypan blue exclusion at the end of PMN SO release assay. Data were analyzed using ANOVA Fisher’s PLSD post-hoc test.

Results:
NTI significantly decreased PMN SO release at 200 μM (n=10, 0.196±0.06, p<0.05) compared to vehicle control (n=22, 0.731±0.07) or NX 100/200 μM (n=3, 0.593±0.06), and NTI at 100 μM (n=7, 0.461±0.08, p<0.05) versus vehicle control. Likewise, KB significantly decreased PMN SO release at 20 μM (n=7, 0.507±0.07), 10 μM (n=7, 0.552±0.07), and 5 μM (n=5, 0.615±0.11) compared to vehicle control (n=10, 0.908±0.07). Cell viability was statistically similar (80-90±5%) among all study groups.

Conclusion:
NTI (10-200 μM) and KB (5-20 µM) exhibited concentration-dependent effects in reducing PMN SO release without compromising cell viability. The results suggest that the novel effect of NTI maybe mediated through a reduction in intracellular Ca
2+
and independent of delta opioid receptor antagonism. These results are consistent with the effects of NTI, KB and NX in myocardial ischemia/reperfusion injuries. Future studies will assess the effects of NTI on ROS attenuation in human umbilical vein endothelial cells.
Abstract We093: Comparative Analysis of Theoretical Models and Fusion Frameworks for Early Cardiac Dysfunction Detection Using Multimodal Wearable Data
Authors: Tawde Poonam
Published: October 2024

Background:
Wearable devices have emerged as promising tools for continuous monitoring of physiological signals, offering opportunities for early detection of cardiac dysfunction. This study aims to compare various theoretical models and fusion frameworks to assess their effectiveness in early detection of cardiac dysfunction.

Hypothesis:
The study investigates the performance of signal processing-based, machine learning-based, and hybrid models for early cardiac dysfunction detection using multimodal wearable data. Additionally, it explores the efficacy of feature-level fusion, decision-level fusion, and sensor-level fusion frameworks in integrating information from diverse sensors for enhanced detection accuracy.The primary goal of the study is to identify the most effective theoretical models and fusion frameworks for early detection of cardiac dysfunction using multimodal wearable data. By comparing different approaches, the study aims to provide insights into the strengths and limitations of each method and contribute to the development of reliable wearable-based systems for proactive healthcare interventions.

Methods/Approach:
Three theoretical models—signal processing-based, machine learning-based, and hybrid—were implemented and evaluated for their performance in detecting early signs of cardiac dysfunction. Additionally, three fusion frameworks—feature-level fusion, decision-level fusion, and sensor-level fusion—were investigated to assess their effectiveness in integrating multimodal wearable data.

Results/Data:
The signal processing-based model achieved a sensitivity of 85%, specificity of 82%, and accuracy of 83% in detecting early signs of cardiac dysfunction. The machine learning-based model outperformed with a sensitivity of 92%, specificity of 88%, and accuracy of 90% . The hybrid model yielded intermediate results with a sensitivity of 89%, specificity of 85%, and accuracy of 87% . Among the fusion frameworks, sensor-level fusion demonstrated superior performance with a sensitivity of 94%, specificity of 90%, and accuracy of 92%.

Conclusion:
Machine learning-based approaches showed higher efficacy in early cardiac dysfunction detection compared to signal processing-based methods. Sensor-level fusion exhibited the best performance among data fusion frameworks, emphasising the importance of integrating information from diverse wearable sensors for improved accuracy.
Abstract We095: Using Human Cardiac Organoids to Elucidate the Cardioprotective Potential of Mitochondrial-Targeted Therapies in a Preclinical Model of Diabetic Cardiomyopathy
Authors: Parker Alex , Lees Jarmon , Phang Jack , Song Sonya , Lim Shiang , De Blasio Miles , Ritchie Rebecca
Published: October 2024

Introduction:
Mitochondrial dysregulation is implicated in many complications of type 2 diabetes (T2D). Mitochondrial-targeted therapies SkQ1, AP39 and SS31 limit renal T2D complications. We sought to obtain the first evidence of the cardioprotective potential of SkQ1, AP39 and SS31 in T2D, using a human induced pluripotent stem cell (iPSC)-derived 3D multicellular cardiac organoid model of diabetic cardiomyopathy.

Methods:
Human cardiac organoids (foreskin-2 cell-line) generated from human iPSC-derived cardiomyocytes, cardiac fibroblasts and endothelial cells were subjected to 5.55mM glucose (control) or T2D-like conditions (20mM glucose, fatty acids, endothelin-1 and cortisol) for 4 days with supplementation of either SkQ1 (50nM), AP39 (50nM) or SS31 (1µM) on day 2. On day 4, endpoint parameters were evaluated in cardiac organoids, including contractile function (captured via Olympus IX71), mitochondrial superoxide levels (MitoSOX assay), mitochondrial membrane potential (TMRM assay), and cellular injury (lactate dehydrogenase; LDH). Data are expressed as mean±SEM (fold control) with
n
=4-6 from 2-3 independent experiments. One-way ANOVA with Dunnett`s
post hoc
analysis was performed with P<0.05 considered significant.

Results:
After 4 days, T2D-conditions increased total contraction duration (1.55±0.10-fold control), time-to-peak (1.71±0.11-fold control, indicative of systolic dysfunction) and relaxation duration (1.46±0.13-fold control, indicative of diastolic dysfunction) in cardiac organoids. All 3 functional parameters were attenuated by AP39 (to 1.15±0.06, 1.32±0.09 and 1.04±0.04-fold control, respectively), or by SS31 (to 1.10±0.05, 1.12±0.15 and 1.09±0.12-fold control, respectively; all P<0.05), but not SkQ1. T2D-conditions increased mitochondrial superoxide (2.68±0.16-fold control), which was blunted by all 3 interventions, SkQ1 to 2.00±0.29, AP39 to 1.82±0.16 and SS31 to 1.89±0.17 (all P<0.05). Cellular injury was evident in T2D cardiac organoids (2.07±0.23-fold control), which was again limited by SkQ1 (to 1.06±0.11), AP39 (to 1.18±0.09) or SS31 (to 1.08±0.07), all P<0.05.

Conclusion:
These findings in human cardiac organoids highlight the cardioprotective potential of mitochondrial-targeted therapies to limit T2D-induced cardiac dysfunction, mitochondrial dysregulation and cellular injury, potentially facilitating clinical progression of candidates such as AP39 and SS31 as new therapies for patients with diabetic cardiomyopathy.
Abstract We111: 3D spheroids composed by induced Skeletal Muscle Progenitor Cells and Mesenchymal Stem Cells derived from human Pluripotent Stem Cells can recapitulate embryonic niches in hindlimb ischemia model
Authors: Kim Jinju , Park Jae-Hyun , Choi Yeon-Jik , Park Hun
Published: October 2024

Introduction:
Peripheral artery disease (PAD) is a significant comorbidity in aging population regarding diminished quality of life and increased mortality rates. There are urgent needs for developing advanced therapeutics for functional limb salvage beyond conventional revascularization. We hypothesized that 3D spheroids composed by induced Skeletal Muscle Progenitor Cells (iSKMPs) and Mesenchymal Stem Cells (iMSCs) could recapitulate embryonic niches and provide promising regenerative potentials in hindlimb ischemia (HLI) models.

Methods:
We manufactured scaffold-free, self-aggregated 3D spheroids using microcavity plates with ultra-low attachment surface. 3D spheroids were designed into 3 groups according to the induced cell types derived from Human Pluripotent Stem Cells (hPSCs); 1) iSKMPs only, 2) iMSCs only, 3) iSKMPs + iMSCs mixed. We transplanted 3D spheroids into the ischemic area and compared their therapeutic potentials using Laser Doppler Perfusion Imaging (LDPI) and immunohistochemical staining analysis.

Results:
iSKMPs secreted various angiogenic factors including IGF, HGF and FGF and facilitated tube formation and migration of endothelial cells (ECs). iSKMPs also showed the differentiation potential into myofibers in defined 2D culture condition. During the 28-day follow-up period, the transplantation of 3D mixed spheroids not only increased blood perfusion in ischemia limb, but also significantly reduced amputation rate compared with those of control and other 3D single spheroid groups. Notably, in vivo capacity of muscle regeneration was more potent in 3D mixed spheroid group with higher rate of engraftment, myotube formation, and vascularization. These findings suggested that iSKMPs played a significant role in limb salvage via myogenesis and angiogenesis, and their cell-to-cell interaction with iMSCs within 3D spheroids provided a favorable microenvironment to enhance the engraftment and maturation of iSKMPs in ischemic hindlimb.

Conclusion:
We demonstrated that the transplantation of 3D spheroids composed by iSKMPs and iMSCs could recapitulate developmental embryonic niches and provide potent regenerative potentials in HLI models. These findings provide compelling evidence that this injectable platform of 3D mixed spheroids can be a promising tool to rescue limb amputation in patients with PAD.
Abstract We117: Preclinical Evidence of Long-term Efficacy and Safety of LX2020, an AAV based Plakophilin-2 Gene Therapy, for the Treatment of Arrhythmogenic Cardiomyopathy
Authors: Zhang Jing , Gutierrez Lara Erika Joana , Do Aryanne , Nguyen Lena , Nair Anju , Selvan Nithya , Fenn Tim , Adler Eric , Khanna Richie , Sheikh Farah
Published: October 2024

Arrhythmogenic cardiomyopathy (ACM) is a fatal inherited cardiac disease caused by mutations in desmosomal anchoring genes, with plakophilin-2 (PKP2) being the most frequently mutated gene (70%). While AAV9-PKP2 gene therapy shows promise in mitigating ACM-related deficits in mice, high dose toxicity and organ failure of AAV9 in human clinical trials raises safety concerns. Prior studies showed AAVrh.10 exhibiting preferential cardiac gene expression at lower doses that could potentially be safer. We hypothesized that AAVrh.10.PKP2 (LX2020) could restore PKP2 to scaffold the desmosomal complex and ameliorate cardiac deficits with long-term efficacy at relatively lower doses and safety in non-human primates (NHP). We employed LX2020 to assess its efficacy in severe (PKP2 homozygous (Hom)) and mild (PKP2 heterozygous (Het)) adult ACM diseased mice, as well as safety in NHP at doses from 2E13 to 1E14 vg/kg and up to 12 weeks post-administration. A dose-dependent increase in hPKP2 expression was seen in all models, with rescue of desmosomal/gap junction protein deficits at 2E13 and 6E13 vg/kg doses in PKP2-Het and PKP2-Hom mouse hearts. Long term LX2020 efficacy was observed at the same doses in PKP2-Hom mice with dose-dependent improvements in cardiac function, arrhythmia reduction and histological improvements, such that LX2020 treated PKP2-Hom hearts resembled wild-type hearts, indicating a potential for reversal of late-stage pathology. The extended lifespan was striking with 100% of PKP2-Hom mice surviving using 2E13 vg/kg, completely averting premature death. Safety assessments in NHP also revealed no adverse events up to the highest dose of 1E14 vg/kg. These data support advancing LX2020 as a potentially viable treatment for PKP2 ACM patients, based on safety and long-term efficacy in rescuing desmosomal and cardiac deficits in ACM models at lower doses reported in the field.
Abstract We126: Myocarditis Following mRNA Vaccination for COVID-19 is Mediated by CXCL10 and IFN-γ
Authors: Cao Xu , Nishiga Masataka , Manhas Amit , Caudal Arianne , Mondejar-Parreno Gema , Zhao Shane , Jahng James , Wang Mingqiang , Utz Pj , Nadeau Kari , Wu Joseph
Published: October 2024

Introduction:
mRNA vaccines have shown remarkable efficacy against symptomatic COVID-19, reshaping the pandemic's trajectory. Despite a robust safety profile, ongoing monitoring is crucial due to the rare but concerning myocarditis cases, predominantly affecting males under 30. Symptoms appear around 2.6 days post-vaccination, with chest pain, shortness of breath, and palpitations. Elevated cTnI and ST-segment changes are common, along with late gadolinium enhancement in cardiac MRI. Understanding these rare adverse effects is essential for the expanding scope of mRNA technology, spanning infectious disease vaccines and anti-cancer immunotherapies.

Hypothesis:
Myocarditis linked to COVID-19 mRNA vaccines involves cytokine production from myeloid cells, leading to impaired cardiac function.

Methodology:
a, Reanalysis of proteomics data of healthy and myocarditis individuals following COVID-19 vaccination; b, Macrophages (N = 7 lines) were exposed to BNT162b2 or mRNA-1273, and analyzed by cytokine array and RNA-seq; c, Cardiomyocytes (N = 4 lines) were treated with CXCL10 (1 and 10 ng/mL) and IFN-γ (0.5 and 5 ng/mL), with or without genistein (10 μM), followed by functional assessment, transcriptomic and proteomic profiling; d, Mice (6 per group) were administered with CXCL10 and IFN-γ, with or without genistein pretreatment (7 days). Myeloid cell infiltration in the heart, serum cTnI levels, RNA-seq, and immunoproteasome activity were analyzed.

Results:
Examination of post-vaccination plasma revealed a notable rise in CXCL10 and IFN-γ levels in patients. Similarly, macrophages exposed to mRNA vaccines displayed heightened production of these cytokines. When cardiomyocytes were exposed to these cytokines, they exhibited compromised contractility, increased arrhythmogenicity, and gene expression patterns reminiscent of myocarditis. Genistein, an anti-inflammatory phytoestrogen, demonstrated significant alleviation of these effects, preventing cytokine-induced proteasomal activation and preserving contractile function. Mouse model of cytokine-induced myocarditis showed that genistein substantially reduced cardiac injury markers and immune cell infiltration.

Conclusion:
These findings highlight CXCL10 and IFN-γ as pivotal contributors to myocarditis following mRNA vaccination and suggest genistein as a potential therapeutic intervention to mitigate associated cardiovascular risks.
Abstract We139: 3D Chromatin Architectures and Transcription Regulation in Diabetic Endothelial Dysfunction
Authors: Feng Yuliang , Cai Liuyang , Wang Yigang , huang wei , Jiang Lei
Published: October 2024

Cardiovascular disease is the major cause of mortality in people with type 2 diabetes, primarily due to endothelial dysfunction. Recent studies have suggested that transcriptional reprogramming plays a central role in endothelial dysfunction, but its regulation in the context of diabetes remains poorly understood. In this study, we utilized 3D chromatin mapping analysis (H3K27ac HiChIP) to investigate the transcriptional regulation in aortic endothelial cells (db/db mice) and HUVEC cells treated with high glucose (HUVEC
high-glu
). We found that these cells were characterized by a massive loss of enhancer-promoter loops, which was associated with transcriptional downregulation. Through transcription factor motif scan, ChIP-seq and ATAC-seq analysis, we uncovered that the lost loop anchors are demarcated by reduced JUNB (transcription factor) binding while their chromatin accessibility remains unchanged. Moreover, we demonstrated that overexpression of JUNB significantly restored enhancer-promoter loops lost in HUVEC
high-glu
and ameliorated endothelial dysfunctions both in vitro (cell scratch, Transwell), ex vivo (aorta relaxation/contraction) and in vivo (hindlimb ischemia) assays. We further showed that JUNB protein (but not RNA) expression is reduced in aortic endothelial cells (db/db), HUVEC
high-glu
and primary endothelial cells from diabetic patients. Through RIME (Rapid immunoprecipitation mass spectrometry of endogenous protein) assay, we identified that RBBP6 (RB Binding Protein 6, Ubiquitin Ligase) is a JUNB interacting protein in HUVEC
high-glu
. We further showed that overexpression of RBBP6 downregulated JUNB expression while proteasome inhibitor MG132 treatment largely abolished the inhibitory effect of RBBP6 on JUNB protein levels in normal HUVEC cells, suggesting that RPPB6 promoted JUNB degradation through ubiquitination. Notably, knockdown of RBBP6 in HUVEC
high-glu
significantly upregulated JUNB expression, enhancer-promoter loopings and improved endothelial dysfunction. Overall, our findings provide novel 3D chromatin insights into diabetic endothelial dysfunction and illustrate a model whereby RBBP6-mediated JUNB degradation leads to global loss of enhancer-promoter looping and transcriptional inhibition in diabetic endothelial dysfunction.
Abstract We142: Identification of the Molecular Determinants for the Circadian Regulation of the Human KCNH2 promoter
Authors: Rozmus Ezekiel , Alimov Alexander , Stumpf Isabel , McCarthy John , Delisle Brian , Schroder Elizabeth
Published: October 2024

Background:
KCNH2 encodes a voltage-gated potassium channel (Kv11.1), which conducts the rapid delayed rectifier potassium current (IKr) critical for ventricular repolarization in the heart. Circadian clock factors regulate the expression of the KCNH2 mRNA transcript in the heart. However, the molecular mechanisms for the circadian expression of KCNH2 transcripts have yet to be defined.

Hypothesis:
A conserved cis-regulatory element in the core KCNH2 promoter drives circadian regulation of human KCNH2 (hKCNH2) promoter activity.

Objective:
Identify the molecular determinants of the circadian regulation of hKCNH2 promoter activity and determine the impact of single nucleotide polymorphisms (SNPs) in this region.

Methods:
Real-time bioluminescence (LumiCycle, Actimetrics) and Dual Glo luciferase assays were employed to identify critical cis elements that regulate the circadian expression of the human KCNH2 promoter using promoter-luciferase reporter constructs. We studied several deletion and mutant hKCNH2 promoter reporter vectors transiently transfected in a myogenic cell line (C2C12 cells). Bioluminescence was measured at 10-minute intervals for 7-10 days. Data were analyzed for circadian characteristics (period, phase and amplitude). Mutant constructs included both engineered and constructs containing naturally occurring rare and common SNPs.

Results:
Lumicycle data demonstrated that the hKCNH2 promoter reporter showed robust circadian and overall activity in C2C12 cells. Deletion and mutational analysis of the hKCNH2 promoter-reporter construct identified a highly conserved tandem E-box element within 1 Kb of the exon 1 start site that was critical for circadian and overall regulation of hKCNH2 promoter activity. Cells expressing hKCNH2 promoter-reporter constructs with different SNPs in this tandem E-box element showed SNP-specific effects. Surprisingly, a relatively common SNP decreased hKCNH2 circadian promoter activity by 66% (n = 6, p=0.0049).

Conclusion:
We identified a conserved tandem E-box in the proximal promoter of hKCNH2 Exon1 necessary for circadian and overall hKCNH2 promoter activity. Several SNPs in this region suggested a functional impact that might lead to decreased levels of IKr.
Cerebellar non-invasive stimulation of social and emotional mentalizing: A meta-analysis
Authors: Van Overwalle Frank , Haihambo Naem , Ma Qianying , Li Meijia , Martínez-Regueiro Rocío , Argoub Ines , Firouzi Mahyar , Deroost Natacha , Baeken Chris , Baetens Kris
Published: October 2024

The present meta-analysis investigated the impact of non-invasive stimulation, using transcranial direct current stimulation (tDCS) and transcranial magnetic stimulation (TMS) targeting the posterior cerebellum, on social and emotional mentalizing about others. Prior research has convincingly shown that the posterior cerebellum supports social and emotional cognition. We identified 14 studies targeting the cerebellum with appropriate control conditions (i.e., sham, control site), which exclude general learning effects of the task or placebo effects. The studies included 29 task conditions where stimulation before or during a social or emotional task was applied on healthy samples. The results showed significant evidence that sustained anodal tDCS and TMS generally improved social and emotional performance after stimulation, in comparison with sham or control conditions, with a small effect size. In contrast, cathodal stimulation showed mixed facilitatory and inhibitory results. In addition, short TMS pulses, administered with the aim of interfering with ongoing social or emotional processes, induced a small but consistent inhibitory effect. Control tasks without social or emotional components also showed significant improvement after sustained anodal tDCS and TMS, suggesting that transcranial stimulation of the cerebellum may also improve other functions. This was not the case for short TMS pulses, which did not modulate non-social and non-emotional control tasks. Taken together, this meta-analysis shows that cerebellar neurostimulation confirms a causal role of the cerebellum in socio-emotional cognition, has a small but significant effect on improving socio-emotional skills, and may, therefore, have important clinical applications in pathologies where social and emotional cognition is impaired.
Mean Kärger model water exchange rate in brain
Authors: Jensen Jens H. , Voltin Joshua , Falangola Maria Fatima
Published: October 2024

Intercellular water exchange in brain is analyzed in terms of the multi-compartment Kärger model (KM), and the mean KM water exchange rate is used as a summary statistic for characterizing the exchange processes. Prior work is extended by deriving a stronger lower bound for mean exchange rate that can be determined from the time dependence of the diffusional kurtosis. In addition, an analytic formula giving the time dependence of the kurtosis for a model of thin cylindrical neurites is demonstrated, and this formula is applied to numerically test the accuracy of the lower bound for a range of model parameters. Finally, the lower bound is measured in vivo with diffusional kurtosis imaging for the dorsal hippocampus and cerebral cortex of 8-month-old mice. From the stronger lower bound, the mean KM exchange rate is found to be 46.1 ± 11.0 s-1 or greater in dorsal hippocampus and 20.5 ± 8.5 s-1 or greater in cortex.
Computation With Sequences of Assemblies in a Model of the Brain
Authors: Dabagia Max , Papadimitriou Christos H. , Vempala Santosh S.
Published: October 2024

Even as machine learning exceeds human-level performance on many applications, the generality, robustness, and rapidity of the brain’s learning capabilities remain unmatched. How cognition arises from neural activity is the central open question in neuroscience, inextricable from the study of intelligence itself. A simple formal model of neural activity was proposed in Papadimitriou et al. (2020) and has been subsequently shown, through both mathematical proofs and simulations, to be capable of implementing certain simple cognitive operations via the creation and manipulation of assemblies of neurons. However, many intelligent behaviors rely on the ability to recognize, store, and manipulate temporal sequences of stimuli (planning, language, navigation, to list a few). Here we show that in the same model, sequential precedence can be captured naturally through synaptic weights and plasticity, and, as a result, a range of computations on sequences of assemblies can be carried out. In particular, repeated presentation of a sequence of stimuli leads to the memorization of the sequence through corresponding neural assemblies: upon future presentation of any stimulus in the sequence, the corresponding assembly and its subsequent ones will be activated, one after the other, until the end of the sequence. If the stimulus sequence is presented to two brain areas simultaneously, a scaffolded representation is created, resulting in more efficient memorization and recall, in agreement with cognitive experiments. Finally, we show that any finite state machine can be learned in a similar way, through the presentation of appropriate patterns of sequences. Through an extension of this mechanism, the model can be shown to be capable of universal computation. Taken together, these results provide a concrete hypothesis for the basis of the brain’s remarkable abilities to compute and learn, with sequences playing a vital role.
On the Prototypical Core of Ancient Greek grástis, Latin grāmen and Old English græs
Authors: Ioannou Georgios
Published: October 2024

The present work explores the relevance of prototype semantics in determining genetic relations in Indo-European linguistics. It argues that schematisation as a constitutive process of category formation can be used as a criterion of economy and analytical coherence, when it comes to corroborating assumed phonological correspondences. Thus, it takes the terms grástis, grāmen and græs in Ancient Greek, Latin and Old English, respectively, all roughly meaning “fodder, grass”, and examines the possibility of all three stemming from a common non-propositional prototypical core. The analysis proceeds in the following way: it first tries to reconstruct a schematic prototype for the series of all meanings related genetically to each of the terms, whereas in turn it compares this prototype for each series with that of the rest. It concludes that the image schema of vigorous internal growth is the prototypical core from which all senses emanate in a conceptual network with radial structure.