bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Tu078: Mechanistic basis of protein phosphatase 2A inhibition by I2PP2A dimerization: A key step in βAR resensitization
Authors: Ashok Anushruti , Mohan Maradumane , Stenson Kate , Sun Yu , Naga Prasad Sathyamangla
Published: October 2024

Dysfunction of beta-adrenergic receptor (βAR) is a key hallmark of heart failure, wherein mechanisms of phosphorylation-mediated desensitization of βARs are well understood. However, less is known about dephosphorylation-mediated resensitization of βARs that is regulated by protein phosphatase 2A (PP2A) in heart failure. Our previous studies have shown that agonist stimulation of βAR activates phosphoinositide 3-kinase γ (PI3Kγ) which phosphorylates endogenous inhibitor of PP2A (I2PP2A) on serine residues 9 & 93. Phosphorylated I2PP2A (pI2PP2A) binds to PP2A inhibiting its activity thereby, impairing βAR resensitization. Although structural studies have shown I2PP2A to be a dimer, yet little is understood about the mechanistic basis of PP2A inhibition by I2PP2A. We therefore hypothesized that phosphorylation of I2PP2A promotes dimerization driving robust interaction with PP2A resulting in inhibition of PP2A and impairment in βAR resensitization. To test for dimerization, hemagglutinin (HA)-tagged I2PP2A and Myc-tagged I2PP2A were generated and co-expressed in HEK 293 cells. Immunoprecipitation of HA-I2PP2A led to significant co-immunoprecipitation of Myc-I2PP2A reflecting the ability of I2PP2A to dimerize. To determine whether phosphorylation is key for dimerization, phospho-mimetic mutants of I2PP2A (S9,93A (inactive) or S9,93D (active)) were generated. Immunoblotting of cellular lysates with expression of these phospho-mimetics showed that S9,93D exclusively formed dimers, while S9,93A I2PP2A only formed monomers suggesting that phosphorylation is key facilitator of dimerization. The role of I2PP2A phosphorylation in its dimerization and PP2A interaction with consequences on βAR resensitization will be presented. Determining the mechanistic basis of this understudied pathway is critical as human heart failure is associated with increased PI3Kγ activity, elevated expression of I2PP2A and reduced βAR function.

Key words:
Desensitization, Resensitization, I2PP2A, PP2A, Dimerization
Abstract Tu089: p21 Regulates Hypertrophic Cardiomyopathy Remodeling By Inhibition of Cardiomyocyte Endoreplication
Authors: Pal Soumojit , Glennon Michael , Nixon Benjamin R. , Chetkof Ethan , Shridhar Puneeth , Kathiresan Chintha M. , CLAVERE Nicolas , Banerjee Dipanjan , Becker Jason
Published: October 2024

Background:
We previously discovered that a murine model of hypertrophic cardiomyopathy had increased cardiomyocyte endoreplication and replication stress leading to increased DNA damage. Activation of DNA damage response pathways was associated with increased cardiomyocyte expression of the cyclin-dependent kinase inhibitor p21. However, the role of p21 on pathologic myocardial remodeling in hypertrophic cardiomyopathy is poorly understood.

Methods:
We utilized a murine model of hypertrophic cardiomyopathy that is deficient in cardiac myosin binding protein 3 (Mybpc3
-/-
). We used murine models deficient in p21 (Cdkn1a
-/-
) or overexpressed p21 (Cdkn1a
SUPER
) to determine the role of p21 on hypertrophic remodeling. Heart structure and function were evaluated by echocardiography. Flow cytometry and immunohistochemistry were used to measure cardiomyocyte DNA content and ploidy. Proteomics and biochemical assays were utilized to identify p21 target proteins.

Results:
We observed that there was an increased p21 expression in Mybpc3
-/-
cardiomyocytes during the postnatal day 7 to 25 time interval. Elimination of p21 expression in Mybpc3
-/-
animals (Cdkn1a
-/-
/Mybpc3
-/-
) caused increased myocardial hypertrophy (Table 1). Conversely, selective overexpression of p21 in cardiomyocytes (Cdkn1a
SUPER
/Mybpc3
-/-
/Myh6Cre) led to reduced myocardial hypertrophy (Table 1). We discovered that isolated cardiomyocyte nuclei from Mybpc3
-/-
mice with reduced p21 had increased polyploidy whereas overexpression of cardiomyocyte p21 led to reduced cardiomyocyte polyploidy (Table 1). Using both proteomics and targeted assays we discovered that cardiomyocyte p21 was binding to key cycle regulatory proteins to inhibit cardiomyocyte endoreplication and hypertrophic growth.

Conclusions:
Collectively, our results show that induction of p21 in hypertrophic cardiomyopathy reduces cardiomyocyte growth by inhibiting endoreplication.
Abstract Tu090: P21-activated kinase 1 safeguards cardiac function from impairment during fasting
Authors: Yarbro John , Chong Lionel , Sooknanan Caleb , Mathew Chris , Singh Pritpaul , Kim Michelle , Ahmed Kinza , Francis Rohan , Kobayashi Tamayo , Huang Yuan , Kobayashi Satoru , Liang Qiangrong
Published: October 2024

Excessive calorie intake poses a heightened risk for cardiovascular disease, while caloric restriction and intermittent fasting have demonstrated benefits in enhancing cardiovascular health and improving cardiometabolic risk profiles. Despite this knowledge, the precise mechanisms underlying the cardioprotective effects of these dietary regimens remain inadequately understood. P21-activated kinase 1 (PAK1) is a serine/threonine protein kinase known to play a crucial role in promoting cardiomyocyte survival in response to various stressors. In this study, we aimed to elucidate the role of PAK1 in maintaining cardiac function during fasting, utilizing mice with modified PAK1 activity in the heart. Under normal feeding conditions, heart function exhibited no discernible differences among wild type (WT), transgenic (TG), and knockout (KO) mice, as evidenced by consistent fractional shortening (FS), ejection fraction (EF), E/A ratio, and isovolumic relaxation time (IVRT). After a one-day fast, systolic contractile function remained robust across all groups of mice. However, the E/A ratio decreased in KO mice, and IVRT was prolonged in KO mice, while shortened in TG mice, highlighting the crucial role of PAK1 in maintaining diastolic cardiac function. Upon a two-day fast, systolic function was compromised in both WT and KO mice, whereas TG mice exhibited preserved function, as evidenced by FS and EF measurements, emphasizing PAK1's equal significance in sustaining systolic cardiac function. Furthermore, fasting accelerated mitophagy, a process eliminating dysfunctional mitochondria through lysosomal degradation, as observed by mitochondria-associated LC3-II and a dual fluorescent mitophagy reporter. Additionally, we established PAK1 as a positive regulator of mitophagy, suggesting its potential protective role in the heart during fasting via mitophagy modulation. In summary, PAK1 emerges as a critical mediator of cardiac function during fasting, presenting promising avenues for therapeutic interventions aimed at improving cardiovascular health.
Abstract Tu112: The Outer Mitochondrial Membrane Protein Mtch2 Regulates Cardiac Metabolic Homeostasis and Body Mass
Authors: Stachowski Marisa , Monroe Tanner , Hadhazy Michele , Bomba-Warczak Ewa , Savas Jeffrey , Puckelwartz Megan , McNally Elizabeth
Published: October 2024

Introduction:
MTCH2 is an outer mitochondrial membrane protein insertase and variants in
MTCH2
associate with human heart failure and obesity.

Hypothesis:
Lower levels of MTCH2 predispose the heart to stress.

Methods and Results:
Cardiomyocyte-specific
Mtch2
KO (
cMtch2
KO) mice were generated and develop systolic dysfunction accompanied by increased fibrosis and elevation of heart failure markers (
n
=6). Prior to the development of cardiac dysfunction male
cMtch2
KO mice maintain a leaner body mass compared to controls, consistent with the idea that cardiac MTCH2 drives whole body energy utilization. We hypothesize the increased energetic demand of maintaining cardiac function in younger animals shifts energy expenditure and results in lower fat mass. We found that young, pre-cardiomyopathic
cMtch2
KO mice were sensitized to stress. When young
cMtch2
KO mice were challenged to an acute dosing regimen of isoproterenol, compared to controls, hearts from
cMtch2
KO mice failed to respond with physiological hypertrophy and animals could not maintain body mass (
n
=7). We assessed mitochondrial function in
cMtch2
KO mice. Isolated
cMtch2
KO cardiomyocytes loaded with Mitotracker green and TMRE had reduced mitochondrial membrane potential compared to control (
n
=20 cells/mouse, 3 mice/group). In the presence of glucose, oxygen consumption rates were reduced in cMtch2 KO heart tissue (
n
=6), and targeted metabolomics of cMtch2 KO ventricle exhibited an accumulation of palmitate (
n
=4), indicating
cMtch2
KO hearts do not efficiently utilize glucose and fatty acids. To identify changes in the mitochondrial proteome we performed mass spectrometry on isolated mitochondria from control and
cMtch2
KO mice (
n
=4). Overall, proteins associated with fatty acid beta oxidation and pyruvate metabolism were reduced in abundance in cMtch2 KO hearts compared to control. Additionally, three potential insertase target proteins were reduced, confirming a critical role for MTCH2 in mitochondrial health.

Conclusion:
MTCH2 is vital for maintaining normal mitochondrial function. Reduction of MTCH2 sensitizes the heart to stress, resulting in impaired mitochondrial, sarcomere, and global heart function with effects on body composition.
Abstract Tu123: Redox Biology of the Mitochondrial Protein mitoNEET and Ascorbate
Authors: Velayutham Murugesan , geldenhuys werner , Khramtsov Valery
Published: October 2024

Background:
It has been proposed that mitoNEET is an Fe-S transfer/repair enzyme, playing a role under conditions of oxidative stress. mitoNEET protects mitochondria against oxidative stress in cardiomyocytes. Loss of mitoNEET is linked with age-related heart failure. Oxidized mitoNEET, [2Fe-2S]
2+
, exports Fe-S cluster from mitochondria to cytosol. Redox control of mitochondrion is essential for the proper functioning of the organelle/mitochondria. However, the molecular mechanisms involved in the redox biology of mitoNEET is not fully studied.

Hypothesis:
Ascorbate regulates redox reactions and Fe-S cluster transport function of mitoNEET in the mitochondria.

Aims:
The objective of this work is to evaluate the redox reactions between mitoNEET and ascorbate.

Methods:
Room temperature and low temperature (77 K) electron paramagnetic resonance (EPR) spectroscopy techniques were used to measure the oxidation of ascorbate and reduced mitoNEET, respectively.

Results:
EPR spectroscopic study shows that mitoNEET oxidizes ascorbate to ascorbate radical. The formation of ascorbate radical increases with increasing concentration of ascorbate. The formation of ascorbate radical is decreased in the presence of menadione. Low temperature EPR study shows that one electron reduction of mitoNEET by ascorbate.

Conclusion:
This study suggest that ascorbate is a substrate/cofactor for mitoNEET. Ascorbate regulates the Fe-S cluster transport function of mitoNEET. Ascorbate plays an important role in the redox biology of mitoNEET under physiological and pathophysiological conditions.
Abstract Tu129: Collagen Peptide modulates Enzymatic Activity post-Myocardial Infarction
Authors: Olabiyi Ayodeji , Cakir Sirin Nazan , de Castro Bras Lisandra
Published: October 2024

Matrix metalloproteinases (MMPs) play critical roles post-myocardial infarction (MI) and directly correlate with adverse LV remodeling. Of the MMPs, MMP-9 shows the highest correlation with MI patient mortality. Clinical trials trying to inhibit MMP-9 were unsuccessful or detrimental, mostly because MMP-9 has critical roles post-MI and inhibition results in exacerbation of adverse remodeling. Previously, we reported a collagen peptide – p1159 – to reduce LV dilation and adverse LV remodeling post-MI. Recently, we discovered p1159 acts as an MMP-9 competitive substrate and reduces substrate proteolysis
in vitro
. Herein, we investigated whether p1159 can act as a competitive substrate
in vivo
to reduce proteolysis of endogenous substrates post-MI without inhibiting MMP-9 activity. Also, we compared p1159 actions to those of a MMP-9 inhibitor (MMP-9i). We hypothesized that animals that receive p1159 would show reduced inflammation and adverse remodeling compared to those that received MMP-9i. Adult male and female C57Bl/6 mice (n = 6-8/group) were grouped as follows: saline (control); p1159; MMP-9i, and p1159 + MMP-9i. We performed baseline echocardiography followed by surgery to induce a permanent occlusion MI model. Three hours later, mice received a randomized osmotic mini-pump to continuously deliver p1159 (14μg/day/kg body weight) or vehicle (saline, 0.9%) and/or MMP-9i (10mg/kg body weight, JNJ 0966 by gavage). The LV and plasma were harvested on day 3 (D3) and D5 for downstream proteomics analysis following a final echocardiographic assessment. Infarct sizes were similar between groups, indicating same level of initial injury. Compared to control, p1159 did not inhibit activity or levels of MMP-9, while animals treated with MMP-9i showed significantly reduced MMP-9 activity. As expected, p1159 reduced (p < 0.05) substrate cleavage, as seen by analysis of the LV peptidome, particularly at D3. Substrates that displayed a reduction in proteolysis included fibrinogen, MIF, myosin, titin, and tubulin. These data support p1159 as a competitive substrate of MMP-9 that can modulate its activity without inhibition. Moreover, these results may explain the previously reported effect of p1159 at reducing LV dilation and improving systolic function.
Abstract Tu136: HIF-1α a novel therapeutic target to reduce Cardiac Ischemia Reperfusion Injury during AMI
Authors: Swain Lija , Reyelt Lara , Qiao Xiaoying , Bhave Shreyas , Everett Kay , Mahmoudi Elena , Kapur Navin
Published: October 2024

Acute myocardial infarction (AMI) is a major cause of mortality worldwide. In preclinical models of AMI, using a trans-valvular pump (TVP) to reduce left ventricular (LV) workload for 30 minutes before reperfusion reduces infarct size by 50%. The mechanisms underlying the cardioprotective effect of LV unloading remain poorly understood. Hypoxia inducible factor 1-alpha (HIF-1α) is an oxygen-sensitive transcription factor that activates cardioprotective signaling pathways, improves cell survival and mitochondrial function during myocardial ischemia. We hypothesized that LV unloading limits myocardial injury by activating hypoxia inducible factor 1-a (HIF-1α).

To explore the effect of LV unloading on HIF-1α stabilization during ischemia, we performed left anterior descending artery (LAD) occlusion for a total of 120 minutes in adult swine in the presence and absence of LV unloading and further 180 minutes of reperfusion was performed following 120 minutes of LAD occlusion.

Trans-valvular unloading activation significantly decreased infarct size both during ischemia (IS/AAR: 4.78±4 % vs 1.5±3%) and post reperfusion when compared ischemia alone and IR alone groups (36.04±8.39 % vs 16.02±3.52 %, p<0.0001) respectively. Interestingly, HIF-1α stabilization is significantly reduced with LV unloading during ischemia and significantly increased post reperfusion in the infarct zone of the swine model of AMI. To test whether HIF1a is necessary for cardioprotection with LV unloading, adult swine were treated with intracoronary delivery of the HIF-1α inhibitor (2-methoxyestradiol; 2ME) before reperfusion. Compared to TVP activation for 30 minutes before reperfusion, addition of 2ME abolished the cardioprotective effect of TVP resulting in increased infarct size (16.03% vs 35.86% p<0.03) and decreased HIF-1a stabilization. Stabilizing HIF-1α post-reperfusion by using a well know HIF-1a stabilizing agent Roxadustat, immediately before reperfusion significantly decreased infarct size in both with and without TVP groups compared to IRI alone. Treatment with Roxadustat also preserved mitochondrial function and increased RISK signaling.

We report the novel finding that stabilizing HIF-1a activity before reperfusion plays a critical role in reducing Infarct size in preclinical model of AMI.
Abstract We011: The role of hsa-miR-9-5p in hypercontractility: insights from patients with hypertrophic cardiomyopathy
Authors: Adao Doris , Grothe Daniela , Hauck Ludger , Mirzaei Zahra , Simmons Craig , Billia Filio
Published: October 2024

Background:
Hypertrophic cardiomyopathy (HCM) is an inherited disease characterized by the thickening of the heart muscle. Mutations in contractile proteins, such as myosin binding protein C (
MYBPC3
) and β-myosin heavy chain (
MYH7
), account for over half of patients with familial HCM. HCM is the leading cause of sudden cardiac death among young adults and athletes, with these mutations potentially leading to hypercontractility and arrhythmias culminating in sudden cardiac death. MicroRNAs (miRNAs) are pivotal regulators of gene expression that may influence disease progression. Despite their potential significance, there is a scarcity of published data on miRNA expression in HCM. Induced pluripotent stem cells (iPSCs) have emerged as valuable tools for modeling various cardiovascular diseases, including HCM. However, the miRNA expression profiles associated with HCM remain largely unexplored within iPSC models. Investigating the differential expression of miRNAs in HCM patients offers a promising avenue to identify potential therapeutic targets for individuals affected by this condition.

Methods:
We performed an unbiased screen using a microarray (NanoString Technologies) that contained 798 miRNAs to identify differentially expressed miRNAs in induced pluripotent stem cell (iPSC)-CM and myocardial tissues derived from healthy donors and HCM patients with
MYBPC3
and
MYH7
mutations.

Results:
We identified 5 differentially expressed miRNAs that are significantly downregulated in both mutant
MYBPC3
iPSC-CM and myocardial tissue (miR-9-5p, -4443, -367-3p, -302c-3p, and -129-2-3p) (p ≤ 0.05). In mutant
MYH7
iPSC-CM and myocardial tissue, 3 miRNAs that were significantly downregulated (miR-9-5p, -151a-3p, -129-2-3p), and 1 was significantly upregulated (miR-331-5p) (p ≤ 0.05). KEGG pathway analysis of gene targets using miRTarBase and g:Profiler revealed that miR-9-5p regulates genes in the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance pathway (p = 3.99x10
-9
), which is involved in hypercontractility associated with HCM.

Conclusion:
This work represents the first description of miRNA expression patterns in iPSC-CMs derived from patients with HCM with additional validation from primary myocardial tissue, the current gold standard. We identified the miR-9-5p/EGFR tyrosine kinase inhibitor resistance axis as a potential therapeutic target responsible for mitigating the hypercontractile phenotype exhibited in HCM.
Abstract We012: Sheet like extracellular matrix secreting single cell microvesicles for reprogrammed endothelial cell therapy towards vascular regeneration
Authors: Morris Viola , Lee Sangho , He Simon , Huang Matthew , Chen Dandan , Kim Kyung Hee , Cho Seonggeon , Pencea Laura , Jain Divit , Yoon Young-sup
Published: October 2024

To enhance cell survival and engraftment for cell therapy, cell encapsulated injectable biomaterials have been widely used. However, the challenges were hypoxia and nutrient shortage in the core, inflammatory cell infiltration and limited distribution of cells from injection sites. Encapsulation of single cells in sub-100-micron microgels with higher surface to volume ratios provides more efficient oxygen and mass exchange. However, typical production of single-cell-laden microgels by microfluidics devices results in cell damage and compromised biological properties.

We hypothesized that stimuli responsive amphiphilic copolymer bearing RGD peptides may self-assemble around single cells induced by cell adhesion moieties would result in single cell microvesicles (MV).

A stimuli responsive amphiphilic copolymer GPM (Gelatin Poly glycerol sebacate Methacrylate) was synthesized by grafting hydrophobic synthetic polymer PGS into hydrophilic natural polymer gelatin via methacrylation followed by aza-Michael addition reaction. Single cell microvesicles of endothelial cells (EC-MVs) were fabricated by self-assembly of GPM at room temperature. For in vivo studies, sub-50-micron EC-MVs made of reprogrammed endothelial cells (rECs) were clustered into 200 micron spheres (mGPM) using sodium alginate and injected into a hindlimb ischemia model to investigate cell survival and biological function.

The mGPM microspheres functioned as a barrier from immune cell infiltration and also enhanced cellular distribution. The encapsulated rECs were released over 2-4 wks in vivo and were shown robust engraftment, migration, and survival in limb tissues, promoting blood flow recovery and sustained vascular regeneration over 6 months. Interestingly, in in vitro culture, rEC-MVs secreted sheet like extra cellular matrix (ECM) which appeared to make an important role for vessel formation (neovascularization) in vivo (Figure 1).

The current study clearly demonstrated the favorable biological applicability of GPM single cell MVs toward vascular regeneration by reprogrammed ECs.
Abstract We013: Modeling Sex and Stress using Induced Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Feinstein Samuel , Chirikian Orlando , Pruitt Beth
Published: October 2024

Despite causing 1 in 3 deaths worldwide, cardiovascular disease and the heart have only recently been studied in women. Contrary to previous belief, the female heart is not just a smaller replica of a male heart. Almost all heart diseases show sexual differences in risk, treatment, and symptoms. Even with these differences there is a lack of female models in animal and cellular studies. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are a powerful human model to test baseline and stressed sex differences at the cellular level. We hypothesized that using stressors that mimic disease conditions (drugs, substrate stiffness) in multiple male and female hiPSC-CMs will result in differential sex-based responses.

We ran Traction Force Microscopy on 3 male (n = 424 cells, 3 batches) and 3 female (n = 318 cells, 3 batches) cell lines on 10 kPa polyacrylamide hydrogels. Male cells had 42% higher twitch force (p = 8.8E-6) with 25% higher contraction (p = 1.77E-5) and relaxation (p = 0.0006) velocities. However, female cells had a larger spread area (823 +/- 17.32 µm
2
vs. 756 +/- 21.64 µm
2
, p = 0.0018). To validate hiPSC-CMs as a model we compared preliminary RNA-seq to publicly available Heart Cell Atlas sc/snRNA-seq data sets of primary ventricular cardiomyocytes. They had similar differentially expressed genes (DEGs) as our hiPSC-CMs. Furthermore, DEGs had significantly enriched gene sets including a 25-fold enrichment in hypertrophic cardiomyopathy (FDR = 1.6E-7), 23-fold in dilated cardiomyopathy (FDR = 1.6E-7), and 16-fold in cardiac muscle contraction (FDR = 6.3E-4). We analyzed stiffness by differentiating and culturing hiPSC-CMs on 10 kPa and 100 kPa stiffness hydrogels. Our RNA-seq analysis at day 10 and 30 after differentiation found 7000 DEGs at 10 kPa while only 556 DEGs at 100 kPa. We looked hypercontractility caused stres and performed RNA-seq on day 60 wild type and hypercontractile mutant hiPSC-CMs. The wild type was dosed with positive inotrope levosimendan to induce hypercontractility while the hypercontractile mutant was dosed with negative inotrope verapamil to lower contractility. Hypercontractility had significant upregulation of the cellular response to stress including ER stress, stress-activated MAPK cascades, stress fibers, and cardiac muscle hypertrophy in response to stress. These differences can identify possible gene targets for preventing cardiovascular disease and inform personalized medicine.
Abstract We016: Generative Artificial Intelligence for hPSC-derived Cardiac Organoid Florescence Generation
Authors: Yang Huaxiao , Kumar Reddy Kandula Arun , Phamornratanakun Tanakit , Huerta Gomez Angello , El-Mokahal Marcel , Ma Zhen , Feng Yunhe
Published: October 2024

Background:
Human pluripotent stem cell (hPSC)-derived cardiac organoids (COs) are extensively applied in modeling heart development and disease. COs are daily monitored by bright-field microscopic imaging for tracking organoid differentiation, morphology, and size, however, it does not provide important information on cell distribution and network in COs. Then, fluorescence microscopic imaging is required with extra steps of experiments when multiple hPSC lines are required. Recently, generative artificial intelligence (AI) has been increasingly applied for image colorization, which extends the possibility of colorizing COs with fluorescence from phase/contrast images. While the phase/contrast image is routinely applied and conveniently used in every biomedical lab daily, is it possible to generate accurate and useful fluorescence information or image colorization from the phase/contrast images of cardiac organoids under the same differentiation protocol is crucial for a broader characterization and analysis of hPSC-derived cardiac organoids and further applications?

Hypothesis:
We hypothesize that generative AI will generate accurate fluorescence images from phase/contrast images of COs.

Approach:
1300 images from COs (differentiated from one reporter hPSC line) were used as the training dataset for the Pix2Pix Conditional Generative Adversarial Networks (GANs) model (
Fig.1a
) with the Convolution Block Attention Module (CBAM) in the U-Net generator (
Fig.1b
). Three models were designed: U-Net (Model 1), U-Net generator with CBAM (Model 2), and U-Net with CBAM and Generator Iteration (Model 3). Three different metrics were included: Peak Signal-to-Noise Ratio (PSNR), Structural Similarity Index (SSIM), and Weighted Patch Histogram to evaluate the prediction outcomes.

Results:
The predicted fluorescence images from phase/contrast images of different COs are very similar to the ground truths (
Fig.1c
). The similarities evaluated by all three metrics (
Fig.1d
) have the scores of PSNR over 30, SSIM over 0.92, and a Weighted Patch Histogram score over 0.75, which are the most accurate and similar to the ground truth.

Conclusions:
A generative AI model was established to colorize phase images of COs using cGANs and CBAM, which capture intricate fluorescence details to support the biomedical research of COs with enhanced interpretability and analysis of cardiac organoid morphology and structure in biomedical research and applications.
Abstract We018: The Molecular Mechanism of Coronary Collateral Growth Induced by Repetitive Ischemia by Single Cell-RNA Sequencing
Authors: Shi Jian , Wang Yang , Juguilon Cody , Gadd James , Enrick Molly , Jamaiyar Anurag , Zhang Laura , Dong Feng , Zhang Yanqiao , Yin Liya
Published: October 2024

Coronary heart diseases (CHD) are the leading cause of mortality and morbidity in the United States. A well-developed coronary collateral circulation ameliorates the consequences of CHD, reducing the incidence of sudden death and infarct sizes following coronary occlusion. Stimulation of coronary collateral growth (CCG) is also an alternative therapeutic approach for patients with intractable angina pectoris. The importance of CCG is undisputable, but the process and mechanism underlying the CCG is unclear. We developed a mouse model of CCG by repetitive ischemia (RI) in adults, and the CCG was validated with contrast echo to measure the coronary blood flow 3D images of micro-CT to quantitate the CCG. In this study, we use single cell-RNA sequencing (scRNA-Seq) to analyze the molecular signaling of CCG at different time points during the RI process. We digested the hearts from RI days 5, 10, and 17 and control (naïve) mice, harvested non-cardiomyocytes, performed scRNA-Seq with 10x Genomics and sequenced with Next-Seq 500 (Illumina).The fastq files are converted to a gene count matrix using kallisto|bustools with refdata-gex-mm10-2020-A employed as the reference mouse genome. The Kallisto bustools output is then converted in R to files compatible for uploading to Cellenics, an open-source platform for single-cell RNA-Seq analysis. Dimensionality reduction is done with Principal Components Analysis (PCA) with 30 principal components. We will use UMAP to visualize cell clusters. Cellenics uses scType to annotate the cell populations automatically when building clusters of cell populations. Our preliminary data show dynamic kinetics of non-cardiomyocytes induced by RI over the timeline of CCG and different cell populations of non-cardiomyocytes and EC expression profiling varied in response to RI throughout CCG. We will validate the critical signaling in CCG and explore the potential targets to induce CCG.
Abstract We027: Gut Microbiota Modulation by Immunosuppression and Cardiac Cell Therapy in a Nonhuman Primate Ischemia/Reperfusion Model of Cardiac Regeneration
Authors: Hsieh Patrick
Published: October 2024

End-stage ischemic heart disease necessitates heart transplantation, and emerging cell therapy presents a promising solution to address donor scarcity. Disruption of gut microbiota significantly influences various diseases and treatments, including transplantation. However, the impact of immunosuppression and cardiac cell therapy on gut microbiota remains largely unexplored. To elucidate this relationship, we investigated gut microbiota dynamics in response to immunosuppression and cell therapy in a nonhuman primate (NHP) cardiac ischemia/reperfusion (IR) model, with controlled genetic, dietary, and environmental factors. Immunosuppression enriched anaerobes (Faecalibacterium, Streptococcus, Anaerovibrio, Dialister), increasing gut microbiota diversity. These changes correlated with metabolic shifts towards amino acid metabolism and nucleosides/nucleotides biosynthesis. Combined treatment of human induced pluripotent stem cell (iPSC)-derived endothelial cells (EC) and cardiomyocytes (CM) also increased gut microbiota diversity, with specific genera alterations. The EC/CM co-treatment group displayed gut microbiota resembling the pre-injury group, with host metabolism shifting towards amino acid and fatty acid/lipid biosynthesis post-cell therapy. These observed microbiota changes and metabolic shifts could serve as biomarkers for monitoring cell therapy and immunosuppression outcomes, offering potential therapeutic targets to enhance efficacy.
Abstract We037: GPC3-mediated metabolic rewiring of diabetic mesenchymal stromal enhances their cardioprotective functions via pyruvate kinase M2 activation
Authors: Joladarashi Darukeshwara , Gurrala Charan Thej , Magadum Ajit , Mallaredy Vandana , Cimini Maria , Gonzalez Carolina , Truongcao May , Gibb Andrew , Benedict Cindy , Koch Walter , Kishore Raj
Published: October 2024

Background:
Mesenchymal stromal cells (MSC) represent promising stem cell therapy for treating cardiovascular diseases. Diabetes affects the functional capability of MSC and impedes cell-based therapy. However, the impact of diabetes on MSC myocardial reparative activity, metabolic fingerprint, and the mechanism of dysfunction is still poorly understood. Therefore, our study aimed to understand the molecular basis of diabetes-induced MSC functions and their metabolic mechanism, and potential metabolic reprogramming of diabetic MSC to reverse these dysfunctions may represent a strategy to enhance cell-based therapeutics for myocardial repair in diabetic patients.

Hypothesis:
Our central hypothesis is that increased Glypican-3 (GPC3) mediates diabetic MSC dysfunction and diminishes energy metabolism; metabolic reprogramming of diabetic MSC with GPC3 knockdown restored diabetic MSC reparative and therapeutic activities in post-MI cardiac repair.

Methods:
MSCs were isolated from the bone marrow of mice from db/+ non-diabetic (WT-MSC) and diabetic mice (db/db-MSC) to compare the MSC metabolic profiles and consequent reparative function. We investigated the effect of GPC3 knockdown on db/db-MSC energy metabolism, immunosuppression, angiogenic potential in vitro, and therapeutic potential for ischemic cardiac tissue repair.

Results:
Intra-myocardial transplantation of db/db-MSC into the ischemic myocardium of mice does not confer cardiac benefit post-MI. The seahorse metabolic flux assays and
13
C glucose tracing studies identified defective energy metabolism in db/db-MSC. Furthermore, we found that GPC3, a heparan sulfate proteoglycan, is highly upregulated in db/db-MSC. GPC3 knockdown-db/db-MSC restored their energy metabolism, immunomodulation, and angiogenesis. Intramyocardial injection of GPC3 metabolically reprogrammed-db/db-MSC confers cardiac benefit post-MI.

Conclusion:
Our findings indicate that metabolic rewiring using GPC3 may be used to restore the metabolic state and functions of db/db-MSC for cell therapy applications.
Abstract We039: Adducin Promotes Cardiomyocyte Sarcomere Disassembly
Authors: Xiao Feng , Nguyen Ngoc Uyen Nhi , Wang Ping , Hsu Ching-Cheng , Li Shujuan , Thet Suwannee , Kimura Wataru , Luo Xiang , Lam Nicholas , Menendez-Montes Ivan , Elhelaly Waleed , Singh Rohit , Sadayappan Sakthivel , Kanchwala Mohammed , Xing Chao , Ladha Feria , Hinson Travis , Hajjar Roger , Hill Joseph , Sadek Hesham
Published: October 2024

Introduction:
Recent interest in understanding the cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. Despite advancements, the intricacies of cardiomyocyte mitosis, especially the breakdown of sarcomeres to enable daughter cardiomyocytes' division, remain largely unexplored.

Hypothesis:
This study investigates the role of Adducin in the disassembly of sarcomeres during cardiomyocyte mitosis, hypothesizing that Adducin serves as a crucial regulator in this process.

Goals:
Our objective is to elucidate the function of Adducin in sarcomere disassembly during the mitosis of mammalian cardiomyocytes and understand how this process is regulated.

Methods:
We examined the expression patterns of α/γ-Adducins, particularly in neonatal cardiomyocytes undergoing mitosis. Subsequently, we experimented with the overexpression of various α-Adducin splices and phosphorus in vitro and in vivo, with a keen interest in the phosphorylation effects of α-Adducin on sarcomere disassembly.

Results:
Here, we identify Adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-Adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phosphoforms of α-Adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-Adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Additionally, co-overexpressing this α-Adducin variant with γ-Adducin in adult mice stabilizes the Adducin complex and maintains sarcomere disassembly, facilitated through interactions with α-Actinin.

Conclusion:
The study highlights Adducin, particularly α-Adducin phosphorylated at Thr445/Thr480, as a pivotal regulator of sarcomere disassembly during neonatal cardiomyocyte mitosis. This mechanism is essential for coordinating the cytoskeletal changes necessary for the mitotic process in cardiomyocytes, offering insights into potential therapeutic targets for cardiomyopathy.
Abstract We042: Gelatin hydrogel elasticity influences TBX18-induced pacemaker cell behavior
Authors: Choi Younghwan , Fan Jinqi , Cho Hee Cheol
Published: October 2024

Introduction:
Extracellular matrix (ECM) stiffness plays a role in determining cell behavior during development and maintenance of organ function. Polystyrene dishes typically show an elastic modulus of 1 gigaPa, which is far from the elastic modulus of 10 to 15 kiloPa for myocardium. Gelatin, a derivative of collagen, is a major structural scaffolding in the myocardium including the sinoatrial node.

Hypothesis:
We hypothesized that gelatin hydrogel at varying stiffness serves as a platform to modulate the automaticity of cardiac pacemaker cells.

Methods:
Freshly isolated neonatal rat ventricular myocytes (NRVMs) were cultured as monolayers on regular, plastic cell culture plate or on 3%, 5%, and 10% gelatin hydrogel that represents a stiffness of 1.3 ± 0.3, 6.4 ± 1.5, and 13.6 ± 3.5 kPa, respectively. All cell culture surface was coated with fibronectin. NRVMs were transduced with Adv-TBX18 or Adv-GFP at day 0 (d0).

Results:
TBX18-NRVMs showed significantly more spontaneous synchronous contractions on the gelatin hydrogel (22 ± 12, 29 ± 1, 33 ± 9 on 3, 5, or 10% gelatin, respectively) than on plastic (10 ± 11) at d4. Control, GFP-NRVMs showed few spontaneous synchronous contractions regardless of the cell culture platform (3 ± 4, 2 ± 3, 0 ± 0 bpm at 3, 5, 10% gelatin, respectively; 3 ± 7 bpm on plastic, n = 5 to 6 wells/group) at d4. TBX18-NRVMs exhibited significantly more spontaneous synchronous Ca
2+
transients/min on 10% gelatin hydrogel (33 ± 2) than on 3% gelatin hydrogel (9 ± 2) or 5% gelatin hydrogel (19 ± 8) (n = 3, p<0.05) at d8. Immunostaining revealed that Hcn4 protein expression in TBX18-NRVMs was higher on 10% gelatin than on plastic at d2. The percentage of vimentin+ nonmyocytes in TBX18-NRVMs was higher on 10% gelatin (39.5 ± 2.2 %) compared to either on 5% (26.6 ± 1.7%) or on 3% gelatin (27.5 ± 4.2) at d6.

Conclusions:
Our data indicate that spontaneous and synchronized contractions of TBX18-induced pacemaker cells were significantly higher on gelatin hydrogel with myocardium-like stiffness compared to those grown on plastic culture medium. The data suggest that modulating ECM stiffness may impact the automaticity of cardiac pacemaker tissues, and could serve as a platform to study sinus node dysfunction.
Abstract We045: Development of SA Node Organoids: A Multicellular Approach to Biological Pacemaking
Authors: Won Jihee , Son Young Hoon , Cho Hee Cheol , Park Sung
Published: October 2024

The rising prevalence of sinoatrial (SA) node dysfunction, attributed to the aging population, is emerging as a critical health issue. The SA node, a natural organoid within the heart, comprises the pacemaker cells embedded within a connective tissue matrix, insulated electrically from the surrounding atrial muscle cells. This isolated architecture, along with a heterogeneous cellular composition, is crucial for the SA node's robust pacemaking and electrical conduction. Developing methods to reconstruct this crucial heterogeneous structure could offer a long-term solution for individuals with SA node dysfunction. However, the use of current biological pacemakers, which typically involve a single cell type, has often led to unstable heart rhythms during one-month in vivo integration, challenging their long-term clinical application as biological pacemakers.

To address existing SA node model limitations, we developed a SA node organoid that reproduces the human SA node's multicellular tissue structure. Initially, we developed a protocol for generating cardiac SA node organoids by employing a lentivirus to deliver dox-inducible genes for key pacemaker transcription factors, specifically TBX-18 or Shox-2. We mixed wild-type human-induced pluripotent stem cells (hiPSCs) with those modified to express TBX-18 or Shox-2. These cell mixtures were aggregated, embedded in a diluted Matrigel, and then exposed to a cardiac differentiation protocol, activating TBX-18 or Shox-2 at specific times (D1 or D7) via Dox administration. By D9, these organoids began to exhibit contraction, which lasted beyond 60 days, with notable upregulation of two transcription factors and pacemaker-specific genes following Dox treatment. Subsequently, we integrated the SA node organoid into 2D hiPSC-derived atrial muscle tissue constructs, maintaining geometric isolation within the cardiac tissue to enhance source-sink mismatch. This isolation allowed the SA node organoid to successfully trigger activity in adjacent dormant cardiomyocytes, preserving the rhythm for over 60 days.

These findings could redefine the role of tissue architecture and cell diversity in the SA node's pacemaking function, potentially leading to a high-fidelity, tissue-level biological pacemaker as a new treatment strategy for SA node dysfunctions.
Abstract We051: Chronic mavacamten treatment prevents development of contractile dysfunction in cMyBP-C null engineered heart tissue constructs, but not in constructs carrying the cMyBP-C W792R missense mutation
Authors: De Lange Willem , Gauchan Angeela , Farrell Emily , Ralphe J Carter
Published: October 2024

Hypercontractility, caused by mutations that increase the number of myosin heads in the disordered relaxed (DRX) state, is a prevalent cause of hypertrophic cardiomyopathy (HCM). A subgroup of HCM mutations cause primary hypocontractility, by stabilizing super-relaxed crossbridges (SRX). Mavacamten (mava) directly targets hypercontractility by reducing the number of DRX heads and improves outcomes in ~70% patients with obstructive HCM.

We here assessed the efficacy of chronic mava treatment at preventing phenotype development in human iPSC engineered heart tissue (EHT) in a cMyBP-C null model (-/-), that increases the number of DRX heads, and EHT carrying the W792R (RR) HCM mutation. While the primary contractile defect caused by the W792R mutation is unknown, we postulated that it differed from the cMyBP-C -/- model, as RR mice die prior to weaning while -/- mice do not.

Twitch force (TF) measurements performed on day 7 on control (+/+), -/- and RR EHT show that -/- EHT were hypercontractile while RR EHT were hypocontractile (Fig. 1). Following measurement, EHT from each genotype were either treated with vehicle or 100nM mava and cultured for 21 days. By day 28, TF production was similar between +/+ and -/- vehicle treated EHT while RR EHT remained hypocontractile (Fig. 2). Chronic mava treatment significantly reduced TF production in +/+ EHT but not in -/- or RR EHT (Fig. 2). Consistent with diastolic dysfunction, a hallmark of HCM in humans, day 28 -/- and RR EHT displayed slow relaxation, though the effect was more pronounced in RR EHT (Fig. 3). While chronic mava treatment accelerated relaxation in -/- EHT to levels that were similar to that observed in vehicle treated +/+ EHT, it failed to do so in RR EHT (Fig. 3).

cMyBP-C -/- EHTs initially present with a hypercontractile phenotype, consistent with an increase in DRX crossbridges, while W792R RR EHT present with a hypocontractile phenotype. By 4 weeks in EHT culture, both genotypes present with impaired relaxation, that is more severe in RR EHT. While chronic mava treatment prevents development of slow relaxation in -/- EHT, it failed to do so in RR EHT. These findings suggest that the primary disease mechanism of the W792R mutation is not an increase in DRX crossbridges and that mavacamten may not be an effective treatment for patients carrying this mutation, further highlighting the need for studying the pathomechansims of HCM mutations and the development of novel therapies.
Abstract We070: The role of neutrophil YAP in cardiac inflammation and injury
Authors: Francisco Jamie , Mareedu Satvik , Guan Jin , Sung Eun-Ah , Zhai Peiyong , Hu Che-Ming , Del Re Dominic
Published: October 2024

Background:
There is a growing appreciation that neutrophils demonstrate plasticity, not unlike the macrophage, and can modulate both inflammation and repair following organ injury. Neutrophils are rapidly recruited to the heart after acute myocardial infarction (MI) and participate in cardiac inflammation and remodeling. Nevertheless, mechanisms regulating neutrophil function during heart injury remain largely unknown.

Research Question/Aim:
Previous work demonstrated that Yes-Associated Protein (YAP) regulates macrophage function during heart injury. However, whether YAP regulates neutrophil function in any context, including reperfused MI (I/R), remains unexplored.

Approach:
We generated neutrophil-specific YAP KO mice (MRP8
Cre
) and subjected them to I/R to elicit inflammation and injury. Cell-based experiments were performed using primary isolated mouse neutrophils. We utilized flow cytometry, single-cell RNAseq, and histological and biochemical assays to investigate underlying mechanisms.

Results:
Neutrophils isolated from the blood and heart of WT mice subjected to I/R revealed increased YAP expression. Compared to control mice, neutrophil YAP KO hearts had decreased infarct size after 24 hours reperfusion, and better cardiac function after 2 weeks. Single-cell RNAseq of heart leukocytes indicated suppression of inflammatory, migratory, and superoxide production pathways in YAP KO neutrophils. Characterization of neutrophils by flow cytometry indicated a shift toward reparative polarization in YAP KO mice during early reperfusion. Bone marrow-derived neutrophils (BMDNs) from KO mice had attenuated inflammatory gene expression and ROS production compared to control cells. Pharmacological inhibition of YAP using verteporfin produced comparable results. Ongoing experiments are investigating how YAP modulates neutrophil inflammatory function, and will determine the therapeutic potential of targeted YAP inhibition for I/R injury.

Conclusion:
These results suggest that YAP regulates neutrophil function during I/R injury in the heart and that suppression of neutrophil YAP affords cardioprotection.
Abstract We083: A new mouse model for the study of cardiogenic TFs
Tbx5
,
Gata4
, and
Mef2c
during cardiogenesis
Authors: Leonard Riley , Sweat Mason , Eliason Steve , Zimmerman Kathy , Zhao Yi , Li Xiao , Hatcher Cathy , Weiss Robert , Amendt Brad
Published: October 2024

Congenital heart defects (CHDs) are be caused by mutations in genes that drive cardiac development, such as
Tbx5
,
Gata4
, and
Mef2c
. Cardiac development, and its transcriptional regulators are also regulated by microRNAs (miRs). These small RNAs target the transcripts of genes in numerous cardiac cell types during embryonic development. We and others have shown that the
miR-200
family modulates the transcripts of cardiogenic transcription factors (TFs)
Tbx5
,
Gata4
, and
Mef3c.
However, the relationship between these miRs and cardiogenic TFs during
in vivo
cardiac development is poorly understood. During cardiogenesis,
miR-200
family members are highly expressed (E14.5) but reduced in adults (3mo) (Ct Value:
miR-200a
: 24.3 ± 0.59 v 38.3 ± 0.21;
miR-200c
: 25.0 ± 0.64 v 32.5 ± 0.16). Using our
miR-200
family inhibitor mice models (PMIS), we have found these miRs are required for cardiac development.
PMIS-miR-200
embryos are found with a ventral septal defect and poor ventricle wall development, which is lethal by e16.5. At e14.5,
PMIS-miR-200
hearts have a significant increase in expression of Tbx5, Gata4, and Mef2c compared to Wild-Type. This induced expression of these TFs is seen within CMs of the ventricle at E14.5. snMulti-Omics of WT and
PMIS-miR-200
hearts found a population of CMs enriched in the
PMIS-miR-200
hearts. These CMs are marked by expression of
Tbx5
,
Nppa
, and
Sox5
. RNA velocity analysis found these CMs to be “progenitor-like” and associated with an early pseudotime. Expression of
Tbx5
,
Nppa
, and
Sox5
correlated along the pseudotime with the “progenitor-like” CMs. ATAC-seq showed enrichment of Tbx5 and Mef2c motifs within this new CM cell state. Conclusions: The
miR-200
family is a modulator of cardiogenic TFs expression and activity during development. Inhibition of
miR-200
induces a CM cell state with “progenitor-like” qualities. Future directions will determine the role of
miR-
200 in adult cardiac disease, such as ischemic injury. Our work provides new insights into gene dosage, modulated by miRs, that is required and necessary during cardiac development.