bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Tu097: Titin cleavage induces loss of myocardial tensional homeostasis driving a rapid TGF-beta-independent fibrotic response
Authors: Lopez-Unzu Miguel , Pricolo Maria Rosaria , Silva-Rojas Roberto , Vicente Natalia , Gavilan-Herrera Manuel , Velazquez-Carreras Diana , Morales-Lopez Cristina , Herrero-Galan Elias , Alegre-Cebollada Jorge
Published: October 2024

Background:
Cardiomyocytes (CMs) are subjected to different types of mechanical loads in the myocardium. Depending on its origin these loads can be classified as extracellular (extracellular matrix-ECM towards CMs), intracellular (exerted by the CM contraction machinery, i.e. the sarcomeres) or intercellular (exerted between CMs). In the myocardium, all these loads are actively maintained in tensional homeostasis (TH). In this context, the study of the sarcomere protein titin is essential, because its I-band region is considered as a hub for mechanotransduction, mostly of intracellular mechanical cues.

Research Questions:
It has been shown that mechanical disruption of the ECM affects the myocardial TH, leading to morphological changes inside CMs. However, potential alteration of TH resulting from defective mechanics of CM proteins remains unknown due to the lack of specific tools for
in vivo
studies.

Aims:
Here we explore the
in vivo
effects on myocardial TH of an abrupt tensional unloading of titin.

Methods:
We have experimentally challenged CMs to a tensional unloading by means of titin cleavage
in vivo
using TEVs-TTN mice. In this model, a cassette with a Tobacco Etch Virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (AAV), we were able to cleave titin
in vivo
, ceasing only the mechanical properties of the protein. Samples were analyzed using an array of techniques including histology, immunofluorescence, and bulk and single nuclei RNA sequencing.

Results:
Our results show that a mosaic expression of TEVp, which does not affect cell viability, cleaving no more than 30% of total titin. However, this leads to a fast fibrotic response (less than six days), characterized by an increase in interstitial collagen and a response from cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this ECM response occurs without noticeable activation of the TGFβ canonical pathway.

Conclusion:
Taken together, our results show experimentally for the first time that titin is not only a mechanotransducer of intracellular cues. In fact, mechanical abrogation of the I-band leads to a fast ECM response mediated by cardiac fibroblasts.
Abstract Tu103: SIRT2 Inhibition Decreases Glycolysis and Attenuates Hypertrophic Response in H9c2 Cardiomyocytes
Authors: Ketema Ezra , Ahsan Muhammad , Persad Kaya , Sun Qiuyu , Zhang Liyan , Lopaschuk Gary
Published: October 2024

Introduction:
Myocardial glycolysis increases in hypertrophic and failing hearts. Hyperacetylation also occurs in the failing heart, and many glycolytic enzymes are known to be subject to acetylation. However, it is generally considered that acetylation has inhibitory effects on glycolysis. As a result, it is not clear whether acetylation changes directly contribute to glycolysis changes in cardiac hypertrophy. We therefore determined whether changes in the acetylation of glycolytic enzymes and the activity of the cytosolic deacetylase SIRT2 regulate cardiac glycolysis.

Methods:
Glycolysis rates were directly measured in rat heart-derived H9c2 cardiomyocytes perfused with 5 mM [5-
3
H] glucose, 0.8 mM palmitate, and 4% bovine serum albumin. Before these metabolic measurements, H9c2 cells were treated with either a SIRT2 inhibitor (10 µM AGK2) or a vehicle for 24 hours. In separate experiments, SIRT2 was also knocked down in H9c2 cells using siRNA, followed by glycolysis rate determinations. The impact of SIRT2 inhibition or SIRT2 knockdown on the acetylation status of glycolytic enzyme was also assessed. Furthermore, the effects of SIRT2 inhibition on hypertrophic signalling were assessed by treating H9c2 cells with phenylephrine.

Results:
SIRT2 inhibition markedly decreased glycolysis rates in H9c2 cells compared to vehicle-treated cells (524±108 vs 2631±372 nmol
.
g dry wt
-1.
min
-1
, p<0.05). Similarly, SIRT2 knockdown resulted in a significant reduction in glycolysis rates compared to scrambled siRNA-treated H9c2 cells (745±31 vs 1659±168 nmol
.
g dry wt
-1.
min
-1
, p<0.05). The decrease in SIRT2 was accompanied by an increase in the acetylation status of the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GPDH). Moreover, a trend towards increased phosphofructokinase (PFK) acetylation was also observed in SIRT2 knockdown H9c2 cells compared to scrambled siRNA-treated cells. Lastly, AGK2 treatment also attenuated phenylephrine-mediated hypertrophic responses in H9c2 cells.

Conclusions:
Increased acetylation of glycolytic enzymes is associated with a decrease in glycolysis, and SIRT2 inhibition or deletion in H9c2 cells significantly decreases glycolysis rates and attenuates hypertrophy. SIRT2 may therefore contribute to the increased glycolysis seen in hypertrophy and heart failure.
Abstract Tu109: Sirt1 inhibits the Sub1-induced RNA polymerase II recruitment to metabolic gene promoters during pressure overload
Authors: Oka Shinichi , Zhai Peiyong , Sadoshima Junichi
Published: October 2024

Downregulation of genes involved in mitochondrial energy production is a hallmark of heart failure, which associates with inhibition of RNA polymerase II (Pol II) recruitment to the gene promoters. Sirt1 is a protein deacetylase regulating transcription of metabolic genes. However, how Sirt1 regulates metabolic genes in heart failure is controversial. Here we show that Sirt1 promotes pressure overload (PO)-induced downregulation of metabolic genes possibly through inhibition of the Pol II recruitment. Cardiac-specific Sirt1 overexpression in mice (Tg-Sirt1) promoted PO-induced cardiac dysfunction (Ejection fraction (%): wild type (WT) sham 76, WT PO 68, Tg-Sirt1 sham 76, Tg-Sirt1 PO 48*, p<0.05 vs WT PO) and downregulation of metabolic genes, including Idh3a (relative Idh3a mRNA levels; WT Sham 1.0, WT PO 0.55, Tg-Sirt1 0.98, Tg-Sirt1 PO 0.41, p<0.05 vs WT PO). In cultured cardiomyocytes, Sirt1 overexpression suppressed metabolic enzymes, reporter gene activity driven by endogenous metabolic gene promoters, and mitochondrial respiration. Chromatin immunoprecipitation and
in vitro
DNA binding assays, using 500bp of endogenous Idh3a promoter sequence, showed that Sirt1 inhibits the Pol II recruitment to the promoter. Unbiased screening using mass spectrometry identified Sub1 as a Sirt1 binding protein. Sub1 is an evolutionally conserved DNA binding protein that mediates the Pol II recruitment via the preinitiation complex formation. We confirmed direct binding of Sub1 to the Sirt1 catalytic domain (235-499aa) and Sub1-dependent Sirt1 recruitment to the Idh3a promoter using the
in vitro
reconstitution system.
In vitro
DNA binding assays with the Idh3a promoter sequence demonstrated that Sirt1 inhibits preinitiation complex formation, but not Sub1 recruitment. These results suggest that Sirt1 inhibits Sub1-induced preinitiation complex formation, thereby suppressing transcription of metabolic genes in the failing heart.
Abstract Tu119: The exocyst trafficking complex mediates fuel transporter membrane delivery in cardiomyocytes.
Authors: Schuller Connor , Ha Herena , Nakamura Nicole , Nelson Luke , Tokunaga Darcy , Luo Emi Lin , Schunke Kathryn , Polgar Noemi
Published: October 2024

Introduction:
To maintain homeostasis, cardiomyocytes (CMs) primarily rely on fatty acid β-oxidation for ATP production facilitated by substrate uptake via fuel transporters. The glucose transporter, GLUT4, and fatty acid translocase, CD36, are directed to the cardiomyocyte sarcolemma in response to insulin signaling and AMP-activated protein kinase (AMPK) signaling. In adverse conditions like acute hypoxia and ischemia, these cells undergo a metabolic shift to favor glycolysis. Previous reports showed that under these conditions, CD36 moved away from the sarcolemma as GLUT4 was delivered to the cell membrane, contributing to a shift from fatty acid oxidation to glycolysis. Investigating CD36 and GLUT4 membrane delivery in cardiomyocytes is key to better understanding cardiac energy metabolism under normal and disease conditions. Prior work in adipocytes, and our own studies in skeletal muscle showed that the exocyst trafficking complex is critical for membrane delivery of GLUT4 and CD36 in response to insulin and AMPK. Our objective was to determine if in cardiomyocytes GLUT4 and CD36 trafficking, and subsequent fuel uptake is dependent on the exocyst.

Methods and Results:
We demonstrate that the exocyst is recruited to GLUT4 and CD36-vesicles in cultured H9C2 cells in response to stimuli with proximity ligation assays. Using on-cell Western analysis and fuel uptake assays we also show that the exocyst is essential for induced GLUT4 and CD36 cell surface delivery and subsequent energy substrate uptake, as inhibition of the exocyst attenuates stimulated surface translocation of both fuel transporters and subsequent fluorescent fuel analog uptake.

Our earlier work revealed that protein levels of exocyst subunits are increased in murine heart tissue following myocardial infarction. To examine the role of the exocyst in fuel uptake under ischemic conditions, we used an in vitro model of simulated ischemia, and found similar alterations in exocyst subunit expression in this model at both mRNA and protein levels. This suggests an increased demand for exocyst activity.

Conclusion:
Further research will explore how ischemia impacts exocyst-mediated trafficking and if the exocyst contributes to the glycolytic shift under ischemia in cardiomyocytes.
Abstract Tu122: Induction of Mitochondrial and Endoplasmic Reticulum Stress in Early Response to High-Fat Diet-Induced Hyperglycemia in Mouse Hearts
Authors: Pal Subhankhi , Eminhizer Nicole , ODwyer Kienan , Muthu Sakthijothi , Tran Zinnia , Prabhu Saina , Velayutham Murugesan , Chantler Paul , Sundararajan Venkatesh
Published: October 2024

Background:
Diabetic cardiomyopathy (DCM) is the leading cause of mortality in diabetic patients, accounting for about 80% of the death. Mitochondria and endoplasmic reticulum (ER) play a critical role in cardiomyocyte function, yet their interaction and function in diabetic hearts remains unclear.

Hypothesis:
We hypothesize that mitochondria and ER stress response are essential in maintaining cardiac function and preventing cardiac remodeling during early diabetic insult in the heart.

Methods:
Utilizing 6-month-old male and female C57BL/6 mice, we divided them into groups receiving a standard chow diet (N=8) and a 60% high-fat diet (HFD, N=8) for four months. Post-diet, we conducted glucose tolerance tests to verify hyperglycemia. We analyzed heart samples for mitochondrial stress (LonP1, ClpX, ClpP, Afg3l2, spg7, GRP75) and ER stress (XbP1, IRE1a, CHOP, PDI, Hspa5, Dnajb9, Hsp90) markers, along with mitochondrial biogenesis (mtDNA, TFAM, Mt-ND4, and Mt COII levels), and whole heart homogenate oxidative potential by electron paramagnetic resonance (EPR) spin probe (CMH). Between the groups, a p-value < 0.05 was considered significant calculated by one-way ANOVA.

Results:
HFD significantly increased body weight (p<0.0001) and fasting glucose levels (p<0.0001), confirming hyperglycemia. Among the mitochondrial stress response markers, LonP1 was significantly (p<0.05) elevated in HFD hearts compared to the control. However, the ER stress-associated transcription factors, CHOP and XbP1, were significantly upregulated (p<0.05) in female HFD hearts compared to the control and male groups. Further, EPR analysis showed a significant (p<0.0001) four- and two-fold increase in oxidative potential in the heart homogenate of female and male HFD groups, respectively. However, mitochondrial biogenesis was notably higher, observed only in HFD females compared to other groups.

Conclusion:
Our study indicates that pre-diabetic hyperglycemia enhances gender-specific mitochondrial biogenesis and triggers mitochondrial and ER stress responses, highlighting the importance of mitochondrial-ER interaction in early DCM and suggesting potential therapeutic avenues.
Abstract Tu126: Unveiling the Role of GRAF1 in Orchestrating Mitophagy and Metabolic Flexibility in Stressed Cardiomyocytes
Authors: Zhu Qiang , Combs Matthew , MacK Christopher , Taylor Joan
Published: October 2024

Mitochondria are essential for cardiomyocyte contraction by generating ATP and orchestrating metabolic pathways. Understanding how mitophagy—selective removal of damaged mitochondria in lysosome—and metabolic flexibility are coordinated is crucial for cardiac function under physiological and pathological conditions. Our study explores the molecular mechanisms behind this coordination in cardiomyocytes.

We identify GRAF1(Arhgap26) as a pivotal mediator in PINK1-Parkin dependent mitophagy, facilitating damaged mitochondria clearance and regulating mitochondrial substrate flexibility. Depletion of GRAF1 in neonatal rat ventricular cardiomyocytes (NRVCMs) compromises mitochondrial function, evidenced by reduced membrane potential, impaired oxidative phosphorylation, and elevated cleaved Caspase 3 levels. Moreover, GRAF1 depletion attenuates LC3II activation and increases mitochondrial mass following toxin-induced stress. Using tamoxifen-inducible cardiomyocyte-restricted GRAF1 knockout mice (GRAF1
CKO
), we observe normal cardiac function under basal conditions. However, upon isoproterenol (ISO) treatment, GRAF1
CKO
mice display cardiac dysfunction and reduced mitophagy. Metabolomics analysis reveal distinct metabolic signatures between ISO-treated control and GRAF1
CKO
mice, with impaired fuel flexibility observed in GRAF1
CKO
mice, as highlighted by impaired glucose utilization relative to control mice. Mechanistically, phosphorylation of specific sites (S668, T670, and S671) within the proline-rich region of GRAF1 by PINK1 or PINK1-dependent kinases regulates its dual functions. This phosphorylation event releases the autoinhibitory state of the SH3 domain, enabling it to interact with ABI2 and WAVE2 complex for actin remodeling necessary for efficient mitochondria clearance. Additionally, phosphorylation enhances GRAF1’s affinity for Malonyl-CoA Decarboxylase (MCD), promoting MCD degradation and subsequent elevation of cellular malonyl-CoA levels. This rise in malonyl-CoA inhibits carnitine pamitoyl transferase-I (CPT-I), thereby suppressing fatty acid oxidation while promoting glucose oxidation in mitochondria, contributing to the mitochondrial metabolic flexibility.

In summary, our findings elucidate GRAF1's role in orchestrating mitophagy and metabolic flexibility in stressed cardiomyocytes. GRAF1 phosphorylation serves as a molecular switch, ensuring efficient mitochondrial clearance and substrate utilization to maintain cardiac energetics and function.
Abstract Tu138: Tandem Mass Tagging (TMT)-based Identification of Proteome Signatures for Ischemia-Reperfusion Injury in Swine
Authors: Sunny Sini , Sun Jiacheng , Aniqa Sayed , Walcott Gregory , Zhang Jianyi , Namakkal-Soorappan Rajasekaran
Published: October 2024

Background:
The composition of proteins in blood plasma mirrors the physical changes occurring in the course of disease pathogenesis. Currently available clinical markers are insufficient to predict the actual damage and the redox changes in response to ischemia. Hence, we identified novel redox markers for ischemia/reperfusion (I/R) in swine using TMT-proteomics.

Methodology:
We employed TMT-10plex isobaric labeling-based quantitative proteomics to track the plasma proteome in Sus Scorfa pigs subjected to surgical I/R at different time points. Plasma samples were collected before ischemia, at 1-hr after ischemia (baseline), and 4-hr, 24-hrs, and 7 days following reperfusion.

Results:
An average of 4853 spectra and a total of 457 proteins were identified, ensuring a 1% false discovery rate for peptides and proteins with at least two distinct peptides. Pre-ischemia plasma samples served as the control group in all the analyses. TMT analysis revealed temporal changes in the plasma proteome with a prominent shift at 4 hours after I/R insult. To investigate the significantly changed proteins (>1.5 fold), a temporal analysis was carried out. Notably, endopin-1, a serpin protein family member, and fibronectin B were detectable in the plasma following 4 hours of ischemia-reperfusion, indicating the release of these proteins in the circulation during the ischemia-reperfusion. A positive correlation was observed between the redox proteins (i.e., catalase and glutathione peroxidase) with the serpin proteins in the plasma. Biochemical analysis of circulating glutathione, a small-molecular antioxidant, revealed a reductive redox after 60 minutes of Ischemia (hypoxic condition) but this was shifted to an oxidative milieu at 4hrs following reperfusion (hyperoxia) in the adult pigs subjected to I/R insult. Interestingly, we noticed a significant increase in GSH levels and a decrease in catalase levels at 24 hours following I/R, suggesting a compensatory biochemical response in association with repair after I/R insult/injury in the heart. These results correlated with the increased circulating LDH levels indicating the rate of tissue damage following I/R.

Conclusion:
The present study reveals the presence of a previously unexplored Serpin protein member, endopin-1, during I/R. Understanding the kinetics of endopin-1 expression along with the shift in redox state can help unravel its role in the progression and resolution phases of I/R injury.
Abstract We002: A natural loss-of-function deletion of the cytohesin 1 (Cyth1) gene in BALB/cByJ mice does not impact cardiomyocyte polyploidy
Authors: Song Ruolan , Sucov Henry
Published: October 2024

The adult mammalian heart has limited regenerative capacity due in part to the low level of diploid cardiomyocytes (CMs). Multiple polymorphic genes determine the final level of diploid CMs, however, the relevant genes need to be identified. There is a large variation in CM ploidy between the highly related sister strains BALB/cJ and BALB/cByJ. We undertook an RNA-Seq comparison of neonatal ventricular heart tissue from BALB/cJ and BALB/cByJ to identify gene expression differences that might underlie this difference in CM ploidy. Cytohesin-1 (Cyth1) and Dynein heavy chain 17 (Dnah17) were particularly different in expression: in BALB/cJ (as in most other mouse strains), Cyth1 is expressed in the heart and Dnah17 isn’t expressed, whereas the opposite profile (Cyth1 off, Dnah17 on) was observed in BALB/cByJ heart. These two genes are immediately adjacent to each other in the mouse genome. We found that the basis of differential expression is a large deletion in the locus of BALB/cByJ mice spanning from the Cyth1 first intron to shortly downstream of the last Cyth1 exon. This creates a complete null Cyth1 allele and at the same time results in ectopic transcription of the downstream gene Dnah17, although this transcript is unlikely to encode a protein. Cyth1 encodes a guanine nucleotide exchange factor that was a plausible candidate for being involved in CM ploidy. Using the natural Cyth1 mutant locus plus an engineered null allele, we found no difference in CM ploidy in the absence of Cyth1 function on a cJ-cByJ F1 background or on an inbred C57BL/6J background. This study successfully identified a natural loss-of-function variant in the Cyth1 gene between BALB/cJ and BALB/cByJ but showed that this does not impact CM ploidy, at least by itself. It may underlie other phenotypic differences between these two strains.
Abstract We020:
Myh9
plays a vital role in cardiac myofibroblast differentiation and is indispensable for cardiac repair after myocardial infarction
Authors: Wang Leshan , Li Yuxia , Liu Qianglin , Zhang Xujia , Adelstein Robert , Fu Xing
Published: October 2024

After myocardial infarction (MI), quiescent cardiac fibroblasts (CFs) are activated and differentiate into myofibroblasts featured by elevated expression of smooth muscle alpha-actin (SMαA, encoded by
Acta2
), which form contractile stress fibers. However, compared to the matrix formation function of cardiac myofibroblasts, much less is known about the role of their contraction in post-MI cardiac repair. In our previous study using mice lacking
Acta2
in CFs, all other 5 actin isoforms were activated to compensate for the loss of
Acta2
, suggesting the low feasibility of abolishing cardiac myofibroblast contraction through deleting actin genes. An alternative strategy through targeting myosin was explored. Our transcriptomic study identified
Myh9
and
Myh10
[encoding non-muscle myosin IIA (NM IIA) and NM IIB, respectively] as the dominant myosin isoforms in cardiac myofibroblasts.
In vitro
studies found that
Myh9
knockout (KO) but not
Myh10
KO significantly affected cardiac myofibroblast morphology, completely abolished the stress fiber formation and contractility of these cells, and reduced their proliferation and ability to stabilize the surrounding matrix. Mice lacking
Myh9
in CFs have an increased post-MI cardiac rupture rate compared to WT mice regardless of sexes (
p
=0.004 and 0.02 for male and female mice, respectively).
In vitro
and
in vivo
transcriptomic and proteomics analysis revealed disrupted expression of stress fiber and mesenchymal identity genes/proteins caused by
Myh9
KO. Mechanistically,
Myh9
KO led to an increased globular to filamentous (G/F) actin ratio. The altered actin dynamics likely trigger multiple epigenetic events, which together lead to significantly altered cardiac myofibroblast gene expression and activities beyond contraction.
Abstract We021: Connexin 43 mediates macrophage induced cardiomyocyte proliferation and heart regeneration
Authors: Li Yandong , Feng Jie , Zhang Jian , Nie Yu
Published: October 2024

Background:
Heart regeneration is a potential strategy to recover pumping function after cardiac injury and protect against heart failure. Macrophages have been demonstrated to promote heart regeneration, while its mechanism has not been addressed. Modulating the interaction between macrophages and cardiomyocytes through connexin 43 (CX43) may provide a clinical therapy for preventing further deterioration of patients with heart injury and restoring the lost myocardium.

Hypothesis:
We hypothesize that macrophages can interact with cardiomyocytes via CX43, leading to promotion of cardiac proliferation and heart regeneration.

Aims:
Our study aims to provide the evidence of macrophages contacting cardiomyocytes through CX43 and promoting heart regeneration.

Methods and Results:
Through cell co-culture experiments, we found that macrophages attache to cardiomyocytes via CX43, subsequently promoting myocyte proliferation as evidenced by proliferative markers. Blocking the CX43 connection by SiRNA between two cells suspended cardiomyocyte proliferation in vitro. We generated macrophage-specific CX43 deficient mice (CX43
Mφ/-
) by crosing CD11b-Cre mice and CX43-flox mice. We performed apical resection operation on 1 day-old CX43
Mφ/-
mice and harvested the hearts at 21 day-post-resection. CX43
Mφ/-
mice demonstrated either the heart regeneration ability nor pumping function. Applying single-cell RNA sequencing in CX43
Mφ/-
hearts, we illustrated the unique transcriptional signature of macrophage and cardiomyocyte in macrophage-specific CX43 deficient mice.

Conclusion:
Our study provides the evidence that macrophages attache to cardiomyocyte via CX43 after cardiac injury, promote cardiomyocyte proliferation and heart regeneration. This discovery may offer a new target for clinical treatment of myocardial infarction.
Abstract We025: Human Engineered Heart Tissues Demonstrate Clinically-Relevant Disease indicators of Duchenne Muscular Dystrophy
Authors: Luttrell Shawn , Arefin Ayesha , Macadangdang Jesse , Gray Kevin , Kharoufeh Samir , Worthen Christal , Luerman Greg , Geisse Nicholas
Published: October 2024

Accurate in vitro modeling of healthy and disease conditions is crucial for assessing the safety and efficacy of novel therapeutics before clinical trials. For cardiac diseases, direct evaluation of muscle contraction is a reliable indicator of overall tissue function, however, this often necessitates expensive and low-throughput non-human
in vivo
models. Moreover, animal models may not accurately predict human responses to compound exposure.

Here, we have developed a turnkey platform for facile fabrication of 3D engineered heart tissues (EHTs) that enables label-free, push-button measurements of contractility. The system allows for customized stimulation protocols to individual constructs, while simultaneously measuring contractility across 24 tissues in parallel with minimal user input. This approach enables stratification of muscle phenotypes and facilitates compound safety and efficacy screening.

We present a 3D model of Duchenne muscular dystrophy that displays functional deficits consistent with in vivo disease indicators, including reduced contractile force and slowed relaxation kinetics, though beat rate remains unchanged. We show both acute and chronic effects of cardiotoxic drugs, as well as positive inotropic responses to external calcium and Isoproterenol. This highlights the utility of using EHTs to identify toxicity and derisk drug development and clinical testing outcomes. Spontaneously beating EHTs were also subjected to in situ stretching, leading to increased levels of cardiac troponin I (cTnI) in the tissue medium, indicating stretch induced injury.

These data demonstrate a first-and-only commercial platform that integrates individual, fully-customizable, well-based control of electrical stimulation across a 24-well plate of tissues to pace muscle constructs, model force-frequency, and enable exercise regimens or damage protocols. Heart organoids were stretched under load presenting a challenge for therapeutics targeting injury prevention in the myocardium. Stimulation is coupled with automated assessment of 3D muscle contraction, providing an inclusive, medium-throughput platform for studying muscle-related disorders, advancing therapeutic discovery using a modality-agnostic biosystem.
Abstract We026: Foxk1 and Foxk2 Promote Cardiomyocyte Proliferation and Heart Regeneration
Authors: Cai Dongcheng , Liu Chungeng , Li Haotong , Nie Yu
Published: October 2024

Background:
The adult mammalian heart exhibits limited regenerative capacity due to restricted cardiomyocyte (CM) proliferation following injuries like ischemia. Foxk1 and Foxk2, members of the Foxk family, are pivotal in cell proliferation and metabolic regulation. However, the roles of Foxk1 and Foxk2 in CM proliferation and heart regeneration remain elusive.

Hypothesis:
We propose that Foxk1 and Foxk2 are master regulators of CM proliferation and heart regeneration.

Methods:
Three genetic mouse lines,
Myh6-CreER
,

Foxk1
fl/fl

, and

Foxk2
fl/fl

were used to generate CM-specific
Foxk1
or
Foxk2
deficient mice. Myocardial infarction was performed in neonatal and adult mice. Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq), RNA sequencing (RNA-seq), qRT-PCR, Western blotting, immunostaining, and Seahorse metabolic assays were conducted to investigate the intrinsic mechanisms by which Foxk1 and Foxk2 regulate CM proliferation and heart regeneration. Masson’s staining and echocardiography were conducted to evaluate cardiac fibrosis and function, respectively.

Results:
Our results showed that the expression of Foxk1 and Foxk2 gradually decreased in CMs during postnatal heart development. CM-specific knockout of
Foxk1
or
Foxk2
inhibited neonatal heart regeneration after MI. AAV9-mediated CM-specific overexpression of Foxk1 or Foxk2 prolonged the postnatal proliferative window of CMs in mice. AAV9-mediated overexpression of Foxk1 or Foxk2 in adult mice improved heart regeneration by promoting endogenous CM proliferation after MI. Through joint analysis of RNA-Seq and CUT&Tag-seq, we found that Foxk1 and Foxk2 regulated
Ccnb1
and
Cdk1
transcription respectively. Knockdown of
Ccnb1
hindered CM proliferation induced by Foxk1 overexpression, and the same effect was observed when
Cdk1
was knocked down in Foxk2-overexpressing CMs. Additionally, Foxk1 and Foxk2 induced CM metabolic reprogramming, suppressing mitochondrial oxidative phosphorylation (OXPHOS) via Hif1α upregulation and reducing oxidative DNA damage, thereby modulating the cell cycle.

Conclusions:
Foxk1 and Foxk2 promote CM proliferation and heart regeneration. Mechanistically, Foxk1 and Foxk2 promote CM proliferation via dual effects that directly promote cell cycle gene transcription and induce metabolic reprogramming. Our findings suggest these transcription factors as potential novel targets in cardiac regeneration post-injury.
Abstract We034: Hjurp Promotes Cardiomyocyte Proliferation and Heart Regeneration by Mediating CenpA Assembly
Authors: Li Haotong , Nie Yu , Hu Shengshou
Published: October 2024

Background:
Loss of cardiomyocytes without replenishment is one of the main cellular mechanisms leading to heart failure. Promoting cardiomyocyte proliferation and heart regeneration has become a therapeutic strategy to rescue heart failure. Therefore, we need to understand the differences between neonatal cardiomyocytes and adult cardiomyocytes, summarize the mechanism of cardiomyocyte exit from the cell cycle, and find potential intervention targets.

Method:
Combined analysis of bulk RNA-seq, ATAC-seq, and histone CUT&Tag of 1-day-old mouse and 7-day-old mouse cardiomyocytes to screen for
Hjurp
.
In vitro
, siRNA was used to knock down
Hjurp
in neonatal rat cardiomyocytes, and adenovirus was used to overexpress
Hjurp
. RT-qPCR and immunofluorescence were used to detect cardiomyocyte proliferation and cardiomyocyte number.
In vivo
, adenovirus was injected into the mouse heart
in situ
to knock down
Hjurp
. Immunofluorescence was used to detect cardiomyocyte proliferation. Masson staining was used to detect the proportion of myocardial fibrosis and echocardiography was used to detect cardiac function to reflect cardiac regeneration and repair.

Results:
HJURP was identified by RNA-seq, ATAC-seq, and histone CUT&Tag as a key regulator of cardiomyocyte proliferation during neonatal heart regeneration in mice. The expression levels of
Hjurp
and
Cenp-A
decrease with the weakening of cardiomyocyte proliferation ability.
In vitro
,
Hjurp
overexpression promoted whereas
Hjurp
knockdown attenuated cardiomyocyte proliferation.
In vivo
, cardiomyocyte proliferation and heart regeneration were suspended in neonatal mice after cardiac injury when
Hjurp
was knockdown in cardiomyocytes.

Conclusion:
Our study confirms that HJURP is necessary for cardiomyocyte proliferation and cardiac regeneration after cardiac injury. As an assembly element of centromeres during the cell cycle, overexpression of HJURP is sufficient to promote cardiomyocyte proliferation, which may be accomplished by promoting CENP-A assembly. Thus, HJURP might serve as a potential novel target in heart regeneration after myocardial infarction.
Abstract We044: MATURITY OF HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES PROMOTED BY BRACHYURY PRIMING
Authors: Kargaran Parisa , Garmany Armin , Garmany Ramin , Stutzman Marissa , Sadeghian Maryam , Ackerman Michael , Behfar Atta , Terzic Andre
Published: October 2024

Introduction:
Cardiac differentiation protocols enable derivation of cardiomyocytes (CM). from human induced pluripotent stem cell (iPS). However, limited tools are available to ensure enhanced iPS-CM maturation without the need for prolonged and complex culture.

Research Question:
This study assessed whether the mesodermal and cardiopoiesis inductor, Brachyury (T), complements standard cardiac differentiation and augments structural and functional maturation of iPS-CM.

Aims:
The impact of T mRNA introduction prior to initiation of cardiac differentiation was characterized on: i) cardiac transcription factor expression; ii) structural and functional hallmarks of maturity; and iii) cardiotoxicity.

Methods:
RT-qPCR was employed to monitor the expression of cardiac-specific transcription factors. Transmission electron microscopy enabled the assessment of cellular ultrastructure. Seahorse measurements were used to determine basal respiration. Fluo4 staining was used to measure calcium transients with external field stimulation. Cardiotoxicity was measured as the impact of doxorubicin on calcium handling and cell survival.

Results:
Transfection with T prompted early and sustained upregulation of the cardiac transcription factors Nkx2.5 (10-fold; FDR<0.001) and MEF2C (7.2-fold; FDR<0.001). Mitochondrial area was increased with T transfection (P<0.001) and T-transfected iPS-CM exhibited greater basal respiration (FDR<0.05), greater calcium handling resilience in response to field stimulation, and a more mature transient morphology. Following doxorubicin exposure T transfected iPS-CM exhibited a greater compromise in calcium handling (FDR<0.001), and exaggerated cell death.

Conclusion:
Induction of human iPS with T prior to initiation of cardiac differentiation enhanced signatures of structural and functional maturity, and provided more robust readouts of drug toxicity.
Abstract We055: Role of Classical Dendritic Cells in the Sterile Inflammatory Response Following Myocardial Infarction
Authors: Katsnelson Michael , Ismahil Ameen , Prabhu Sumanth
Published: October 2024

Myocardial infarction and the subsequent development of chronic ischemic cardiomyopathy is the most common cause of congestive heart failure. Infarction is associated with an intense sterile inflammatory response which can lead to adverse remodeling over the long term. Dendritic cells (DCs) are antigen presenting cells that function as a bridge between innate and adaptive immunity. Precursor DCs give rise to several distinct classical DC (cDC) subsets that mediate a variety of functions. The cDC1 subset is essential for cross-presentation of antigens to CD8
+
T cells, while the cDC2 lineage is a potent inducer of CD4
+
T cell proliferation. The aim of the study was to characterize the profile of the cDC populations present during the various stages of the development of chronic ischemic cardiomyopathy. We
hypothesized
that cDCs are essential mediators of chronic LV remodeling in the period following ischemic myocardial injury. We sought to determine which specific cDC subtype (cDC1 vs cDC2) forms the predominant population in the heart and secondary lymphoid tissues following ischemic injury. Adult male C57BL/6 (WT) mice underwent non-perfused myocardial infarction by ligating the left anterior descending artery (LAD) to induce sterile injury, with sham operated mice used as controls. Left ventricular (LV) remodeling was assessed by echocardiography at 1 w and 8 w following surgery. Alterations in cDC1 (CD45+CD11c+CD103-XCR1-) and cDC2 (CD45+CD11c+CD103+XCR1+) profile were examined by flow cytometry. When compared with the sham operated mice, mice with ischemic cardiomyopathy following LAD ligation exhibited the following features. There is an initial influx of cDC2 into the myocardium 1 w after MI (mean of 3.0% vs 0.26% of CD45
+
leukocytes, p=0.02). This is followed by a subsequent influx of cDC1 into the myocardium 4 weeks after MI (mean of 1.98% vs 0.16% of CD45
+
leukocytes, p=0.001). This influx of cDC into the heart is accompanied by an egress of cDC from the spleen (mean of 0.37% vs 0.76% of CD45
+
leukocytes, P=0.001) at 4 weeks following MI. In conclusion, the findings of our study suggest that there is an initial influx of cDC2 followed by a more sustained influx of cDC1 into the myocardium in the weeks following an infarct. Antigen presentation by cDC likely plays a critical role in driving the inflammatory response and inducing chronic LV remodeling in the period following MI.
Abstract We059: Age-associated Alterations in T cell Output and Functions
Authors: Konovalova Daria , Shane Danielle , Mabito Megan , Yuan Sarah , Ma Yonggang
Published: October 2024

The elderly have a higher risk for a wide range of diseases, including infection, tumor, and cardiovascular disease. This could be, at least partially, attributed to T lymphocyte changes during aging. The goal of this project was to characterize age-related changes in T cell output and functions. In this study, young (3-6 months old) and aged (18-24 months old) male and female C57BL/6J mice were utilized. Our data showed that compared to young mice, aged mice demonstrated significantly smaller thymus and fewer developing thymocytes, including double negative CD4
-
CD8
-
, double positive CD4
+
CD8
+
, as well as single positive CD4
+
and CD8
+
thymocytes, which implies that aged mice generate less T cells. Accordingly, circulating T cell count was significantly lower in aged mice than young controls. To investigate how aging affects T cell functions, we isolated splenic T cells and stimulated them with PMA (100 nM) + ionomycin (1 µM) (P+I) or vehicle for 4 hours. At the steady state, aged T cells expressed higher levels of
Ifng
,
Tnfα
,
granzyme b
, and
perforin
than young T cells, suggesting that aged T cells show a senescence-associated secretory phenotype (SASP). After stimulation with P+I, both young and aged T cells expressed dramatically higher levels of
Ifng
,
Tnfα
,
Il2
,
Il4
,
Il17
, and
granzyme b
, indicative of T cell activation. Interestingly, aged T cells had higher expression of
Ifng, Il17,
and
granzyme b,
but lower
Tnfα
and
Il2
levels. Moreover, aged T cells expressed higher levels of exhaustion markers, such as
Pd1
,
Ctla4
,
Lag3
, and
Tigit
, indicating that aged T cells exhibit an exhaustion phenotype. In conclusion, our study demonstrates that aged mice show smaller thymus and fewer thymocytes and blood T cells than young mice. In addition, aged T cells exhibit a SASP and exhaustion phenotype. Upon activation, aged T cells might be more cytotoxic by secreting higher levels of IFN-γ, IL-17, and granzyme B.
Abstract We073: CLEC4E Signaling in Myocardial Ischemia-Reperfusion Injury
Authors: Algoet Michiel , Pusovnik Matic , Caluwe Ellen , Gillijns Hilde , Gsell Willy , Himmelreich Uwe , Oosterlinck Wouter , Janssens Stefan
Published: October 2024

Background:
The innate immune receptor CLEC4E (C-type lectin domain family 4 member E) in cardiac resident cells and circulating leukocytes contributes to ischemic myocardial damage and adverse LV remodeling following ischemia reperfusion injury (I/R). Mice with loss of CLEC4E gene function show reduced cardiac injury and better LV remodeling but the mechanisms of this cardioprotection remain unknown.

Aims:
We investigated whether Clec4e gene function in cardiac resident cells vs infiltrating immune cells mediates cardioprotection during myocardial ischemia-reperfusion injury.

Methods:
We generated chimeric mice by IV infusion of 5 x 10
6
bone marrow cells originating from C57Bl6/J CLEC4E
-/-
and C57Bl6/J wild-type (WT) donor mice in 8w-old lethally irradiated (9.5 Gy) WT (n=36) and CLEC4E
-/-
(n=50) receptor mice, respectively. After 4 w, mice were subjected to ischemia reperfusion injury (I/R) by 60 min LAD occlusion. Troponin levels (TnI) were measured at 90 min post I/R and 7T-magnetic resonance imaging with late gadolinium enhancement (LGE) for infarct size evaluation was performed at 24 h and 28 d post I/R. Mice were euthanized at 28 days for immunohistochemistry and analysis of BM chimerism.

Results:
Immunohistochemistry for CLEC4E expression on bone marrow smears confirmed successful BM chimerism. At 24h post I/R, TnI levels and LGE-assessed infarct size on MRI were not significantly different between groups (42.1 vs 48.4 ng/ml, and 25.2% vs 31.5% of LVmass in CLEC4E
-/-
& WT BM versus in WT & CLEC4E
-/-
BM, respectively) suggesting similar myocardial damage . After 28 d, LV end-diastolic volumes (43µL vs 55µL), and end-systolic volumes (13µL vs 25µL) were significantly smaller in CLEC4E
-/-
& WT BM than in WT & CLEC4E
-/-
BM mice (P<0.05 for both), resulting in better preserved global LV function (LVEF 62% vs 47%, P<0.05) . Infarct size, semiquantitatively assessed using planimetry on Sirius red stains, was smaller in CLEC4E
-/-
& WT BM than in WT & CLEC4E
-/-
BM (6.9% vs 19.8%, P<0.01).

Conclusion:
CLEC4E gene deletion in cardiac resident cells, but not in infiltrating hematopoietic immune cells is associated with better functional and structural remodelling after myocardial I/R. Inhibition of CLEC4E signaling via blocking antibodies or small molecule inhibitors in cardiac myocytes might be a promising cardioprotective strategy.
Abstract We078: Endothelial-specific PHD2 Modulates Cardiac Inflammation, Hypertrophy, and Dysfunction in an Age-dependent Manner
Authors: He Xiaochen , Xu Rui , Bhattarai Umesh , Pan Lihong , Wang Dongzhi , Niu Ziru , Zeng Heng , Chen Jian-Xiong , Chen Yingjie
Published: October 2024

Background:
Prolyl hydroxylase domain proteins (PHD) are oxygen-sensing molecules that degrade hypoxia-inducible factor-α. Deletion of endothelial-specific PHD2 (PHD2ECKO) shows different degrees of pulmonary arterial hypertension and cardiac hypertrophy. Whether EC-specific PHD2 deletion plays a role in regulating cardiac inflammation and dysfunction is still unclear. We investigated the effect of endothelial-specific PHD2 deficiency on cardiac inflammation and dysfunction in mice.

Methods:
Briefly, PHD2ECKO mice were obtained by crossing PHD2-floxed mice with VE-Cadherin-Cre transgenic mice. The effect of PHD2ECKO on cardiac structure and function was determined in male young (6 months old) and old (12-16 months old) mice.

Results:
While PHD2ECKO caused significant left ventricular (LV) hypertrophy in mice at 6 months, PHD2ECKO had no significant impact on LV fibrosis and dysfunction in these mice. However, PHD2ECKO caused significant LV leukocyte infiltration, fibrosis, cardiomyocyte hypertrophy, and LV dysfunction in mice over 12 months. Most interestingly, we found that PHD2ECKO caused a significant increase of cardiac CD4
+
T cells and CD8
+
T cells, activation, and their proinflammatory cytokine productions in mice at the age of 12 months. Moreover, we found that PHD2ECKO resulted in a significant upregulation of several adhesion molecules important for CD4
+
and CD8
+
T cell homing, retention, and proliferation. However, the cytokine production by other immune cell subsets was largely unaffected.

Conclusions:
Our findings demonstrated that endothelial-PHD2 exerts an important role in proliferation, activation, and proinflammatory cytokine production by CD4 and CD8 T cells, and the aging-dependent LV hypertrophy and dysfunction.
Abstract We080: Mapping the effect of Hedgehog signaling on cardiac differentiation and migration in space and time
Authors: Tarampoulous Isaak , Leo Justin Avryn , Ponnuswamy Kirthi , LaRiviere Patrick , Moskowitz Ivan
Published: October 2024

Background:
Organ morphogenesis requires precise control of organ-specific progenitor cell migration and differentiation. Hedgehog signaling is a major signaling pathway during animal development and is essential for proper cardiac development. Previous work demonstrated that Hedgehog signaling can regulate migration and the timing of differentiation of cardiac progenitors. However, how Hedgehog signaling integrates control of differentiation timing and migration in space to enable proper organ formation during development remains unclear.

Hypothesis:
We hypothesize that Hedgehog signaling explicitly controls cardiac morphogenesis by coordinating cellular migration and differentiation of cardiac progenitors to determining where and when they turn into cardiomyocytes.

Methods:
We combine mouse genetics,
in-vivo
lineage tracing, whole mouse embryo light-sheet imaging, and metanalysis of publicly available spatial transcriptomics datasets to address the hypothesis.

Results:
We identified the sites of active Hedgehog signaling by studying a reporter for Hedgehog signaling (GBS-GFP). Next, we determined the necessity of Hedgehog signaling for differentiation of cardiomyocytes at the right place and time by tracing the cardiac progenitors over developmental time in mutants in controls compared to Hedgehog signaling mutants.

Conclusion and future directions:
This work will establish the dual role of Hedgehog signaling in differentiation timing and migration control as a novel paradigm that may be generalizable to other organ systems dependent on Hedgehog signaling (e.g., neural cord and limb patterning).
Abstract We087: The mitochondrial citrate carrier SLC25A1 is a regulator of metabolic reprogramming and morphogenesis in the developing heart
Authors: Ohanele Chiemela , Peoples Jessica , Karlstaedt Anja , Geiger Joshua , Ghazal Nasab , Brown Milton , Davis Michael , Porter George , Kwong Jennifer
Published: October 2024

Background:
Congenital heart defects (CHDs) are the most common type of birth defect, yet the etiology of most CHDs are unknown. A major genetic risk factor for CHD is 22q11.2 deletion syndrome (22q11.2DS), a multisystem chromosomal microdeletion disorder where ~75% of patients present with CHD.
SLC25A1
is a gene found within the 22q11.2DS microdeletion region that encodes for the mitochondrial citrate carrier, an inner membrane transporter that regulates intracellular citrate compartmentalization. Through this role, SLC25A1 is thought to contribute to cytosolic acetyl-CoA availability, and processes like protein acetylation and the epigenetic regulation of gene expression. We have previously found that
SLC25A1
is a central gene contributing to the neurodevelopmental presentation of 22q11.2DS and importantly, neuronal mitochondrial function. Because the heart, like the brain, is highly reliant on energy derived from mitochondria, and the developing heart must undergo metabolic maturation whereby metabolism is reprogrammed from primary reliance on glycolysis towards mitochondrial oxidative metabolism, we hypothesized that SLC25A1 plays a key role in the metabolic maturation that is required for proper cardiac development.

Objective:
Here, we studied the role of Slc25a1 in the developing heart to examine the link between mitochondria and cardiac morphogenesis.

Methods and Results:
By studying mice with the systemic knockout of SLC25A1, we discovered that
Slc25a1
null embryos displayed impaired growth, cardiac malformations, mitochondrial ultrastructural defects and impaired mitochondrial oxygen consumption. Transcriptomics analyses of metabolism-related genes revealed that
Slc25a1
deletion causes widespread alterations in metabolic gene expression. Further, metabolic modelling predicted that loss of SLC25A1 downregulates oxidative phosphorylation, while increasing reliance on glucose. Finally, we found that loss of SLC25A1 decreases cardiac H3K9 acetylation levels at promoter regions of dysregulated metabolic genes. Mechanistically, SLC25A1 may regulating mitochondrial function and metabolism through epigenetic control of gene expression to promote metabolic remodeling in the developing heart.

Conclusions:
Our work suggests that SLC25A1 as an an important mitochondrial regulator of gene expression in the developing heart. Importantly, it positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis as well as cardiac metabolic maturation.