bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1003: Mechanical Loading Controls Cardiomyocyte Proliferation
Authors: Ciucci Giulio , Colliva Andrea , Vodret Simone , Zago Elena , Maglione Manuel , Cimmino Giovanni , Golino Paolo , sinagra Gianfranco , Loffredo Francesco , Zacchigna Serena
Published: November 2022

Introduction:
Mammal cardiomyocytes (CMs) lose their proliferative capacity early after birth and switch to an adult phenotype, characterized by organized sarcomeres. The mechanisms that control this switch remain largely elusive. Among the changes experienced by the heart at birth is a sudden increase in mechanical load.

Hypothesis:
This study tests the hypothesis that variations in mechanical loading may regulate both the proliferation and the maturation of cardiomyocytes.

Methods:
Engineered heart tissues (EHTs) were generated using neonatal rat heart cells, with a modified protocol allowing to alter mechanical load. Unloading is obtained by reducing the distance between the silicon posts that anchor the extremities of the EHT, while overloading is induced by changing Young’s module of posts using metal braces. Adult heart unloading in vivo was achieved by heterotopically transplanting a mouse heart into the neck of syngeneic recipient mice, which were injected with EdU twice per week. Both hearts, native and transplanted, were harvested at day 30.

Results:
In line with our hypothesis, mechanical unloading of developed EHTs significantly increased the percentage of EdU-, Ki67- and pH3-positive CMs at 72 hours. In contrast, increasing afterload induced CM maturation, characterized by increased cross-sectional area and force of contraction, associated with reduced proliferation. Consistently, heterotopically transplanted hearts showed a significant increase in the the number of proliferating CMs in both ventricles, which were almost absent in native hearts.

Conclusions:
Our data indicate that mechanical unloading induce CM proliferation in both rat EHTs and an in vivo in a mouse model of heterotopic heart transplantation. These results support our initial hypothesis and indicate that mechanical loading is a master regulator of cardiomyocytes proliferation and maturation. Ongoing experiments aim at identifying the molecular mechanisms that regulate this process.
Abstract P1004: Macrophage Apoptotic Cell Receptor Mertk Triggers The Synthesis Of Arachidonic Acid Derivatives To Promote Cardiac Regeneration
Authors: Lantz Connor , Becker Amanda , Thorp Edward B
Published: November 2022

Introduction:
Macrophages (MΦs) are indispensable for regeneration of the mammalian neonatal heart. However, the underlying mechanisms by which neonatal MΦs sense the injured cardiac milieu and promote regeneration remain obscure. Utilizing single-cell transcriptomics, we previously observed a regenerative neonatal cardiac MΦ population that distinctly upregulates MerTK, an apoptotic cell receptor, compared to adult cardiac MΦs.

Objective:
We hypothesized that MerTK is required to program neonatal cardiac MΦs to secrete specialized pro-“regenerative” lipids to induce cardiomyocyte proliferation leading to heart regeneration.

Methods:
Postnatal day 1 and adult mice were subjected to cardiac injury via ligation of the left anterior descending artery. Single-cell transcriptomics, lipid-omics, and molecular approaches were utilized to determine the mechanisms by which neonatal MΦs regulate cardiac regeneration.

Results:
To determine the necessity of MΦ MerTK for cardiac regeneration, we employed a myeloid-specific knockout of MerTK (
mMertk
-/-
) which failed to regain homeostatic cardiac function. We next performed single-cell transcriptomics on

Mertk
+/+

and
Mertk
-/-
neonates to determine how MerTK activation reprograms neonatal MΦs for regeneration.

Mertk
+/+

MΦs significantly upregulated genes associated with arachidonic acid metabolism. Hypothesizing that arachidonic acid derivatives may be key signaling molecules for regeneration, we performed a targeted lipid-omics screen which revealed an increase in Thromboxane B
2
(TXB
2
) in regenerating hearts but not within non-regenerative
Mertk
-/-
hearts. Furthermore, addition of a TXA
2
agonist to neonatal cardiomyocytes
in vitro
significantly increases their proliferation by regulating the YAP/TAZ signaling. Alternatively, inhibition of thromboxane synthase in neonatal MΦs or thromboxane receptor on cardiomyocytes by small molecule inhibitors significantly abrogated the increase in cardiomyocyte proliferation by efferocytic

Mertk
+/+

neonatal MΦs.

Conclusions:
Altogether, our data suggest that MerTK programs neonatal cardiac MΦs to secrete TXA
2
to induce cardiomyocyte proliferation via the HIPPO-YAP/TAZ pathway prompting neonatal cardiac regeneration.
Abstract P1007: Cardiac Fibroblast BAG3 Controls TGFBR2 Signaling And Contributes To Engineered Cardiac Tissue Function.
Authors: Wang Bryan Z , Luo Lori J , Zhang Xiaokan , Morsink Margaretha , Soni Rajesh , Lock Roberta I , Kim Youngbin , Nash Trevor , Gorham Joshua , Fine Barry , Seidman Jonathan G , Seidman Christine E , Vunjak-Novakovic Gordana
Published: November 2022

Introduction:
Mutations in BAG3 result in dilated cardiomyopathy with cardiac fibrosis. Currently, the role of BAG3 in non-myocyte cell types is not known. We tested the hypothesis that BAG3 controls cardiac fibroblast (CF) function and that the loss of fibroblast-specific BAG3 is deleterious, using cells and engineered cardiac tissues (ECT) derived from pluripotent stem cells (iPSC).

Methods:
Isogenic iPSCs containing homozygous knockout (KO) of BAG3 were differentiated into CFs. Mass spectrometry measured the effect of BAG3 loss on CF proteome. EdU staining and flow cytometry were used to assess CF proliferation. Transforming growth factor beta (TGF) signaling pathway activity was measured using a luciferase reporter and Western blotting. Affinity purification mass spectrometry (AP-MS) identified high-confidence interactors of BAG3 in CFs. To assess the role of fibroblast-specific BAG3 in cardiac function, conditional KO ECTs were generated using wild-type cardiomyocytes mixed with either wild-type CFs or KO CFs. ECTs were functionally assessed using video microscopy and immunofluorescence. The effects of CF BAG3 loss on tissue gene expression are under study by single nuclear RNA sequencing.

Results:
Analysis of proteomics data revealed enrichment of TGF and proliferation pathways in KO cells. KO-CFs had increased proliferation measured by EdU staining and increased SMAD luciferase reporter activity. Western blotting showed that KO CFs have higher expression of TGF beta receptor 2 (TGFBR2) and activation of canonical TGF signaling. AP-MS identified TGFBR2 as an interacting partner of BAG3, suggesting BAG3 mediated TGFBR2 turnover. In ECT, the loss of CF BAG3 caused a reduction in cardiac tissue contractile force (50% reduction, n = 10-12 tissues per group). Immunofluorescence revealed significantly increased tissue fibrosis in CF-KO cardiac tissues quantified by vimentin staining.

Conclusions:
BAG3 loss results in increased TGFBR2 signaling and proliferation in cardiac fibroblasts. Conditional KO ECTs demonstrate that CF BAG3 affects tissue function. These data suggest that fibrosis related to BAG3 pathology may be a result of a primary deficiency in CFs rather than a secondary response to cardiomyocyte death.
Abstract P1020: Differential Contribution Of Bone Marrow Cells To The Heart In Steady-state And After Repetitive Ischemia
Authors: Shabani Parisa , Shockling Lindsay , Enrick Molly , Kolz Chris , Ohanyan Vahagn A , Yin Liya , Chilian William M
Published: November 2022

Multiple studies have implicated the important role of migration of bone marrow (BM)-derived cells in heart after ischemia. Various BM-derived cells have been demonstrated to exert beneficial or detrimental effects in ischemia. But the response of BM to repetitive ischemia (RI) and the difference in contribution of this heterogenous population to the heart after RI remain elusive. Here, we applied an occluder on the left anterior descending coronary arteries of rats and they went under RI over a period of 10-17 days. This RI model induced coronary collateral growth and increased myocardial blood flow in the ischemic region after 10-17 days. We performed single- cell RNA sequencing and analysis of 24103 bone marrow transcriptomes isolated from the heart and bone marrow of rats in steady-state and after RI. Unsupervised clustering of cardiac neutrophils revealed 28 major clusters and 16 different cell types. Compared to steady state heart, proportion of BM derived macrophage mainly CD163 positive population was increased in the heart after RI. The proportion of BM derived natural killer cells was decreased markedly in the RI group. There was a slightly higher percentage of BM derived endothelial cells and lower smooth muscle cell and fibroblast in the RI group. While neutrophil degranulation, leukocyte activation, leukocyte migration are the major biological pathways in both groups, RI group showed activation of regeneration. We report heterogeneity of BM cells migrated to the heart during RI and redefine the BM that respond to heart ischemia and participate in induction of angiogenesis after RI.
Abstract P1021: Translational Landscape Of Direct Cardiac Reprogramming Reveals A Role Of Ybx1 In Repressing Cardiac Fate Acquisition
Authors: Xie Yifang , Yang Yuchen , Liu Jiandong , Qian Li
Published: November 2022

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise for heart regeneration. Although great progress has been made in understanding the transcriptional and epigenetic mechanisms of iCM reprogramming, its translational regulation remains largely unexplored. Through integrative ribosome and transcriptomic profiling, we characterized the translational landscape of iCM reprogramming, and found extensive translatome repatterning during fibroblast conversion into iCM. Loss of function screening for translational regulators uncovered Ybx1 as a critical barrier to iCM induction. Knockdown of
Ybx1
dramatically improved the efficiency and quality of iCM reprogramming. Mechanistically, depletion of
Ybx1
de-repressed the translation of its direct targets
Srf
and
Baf60c
, both of which mediated, at least partially, the effect of
Ybx1
depletion on iCM generation. Interestingly, upon removing Ybx1, Tbx5 alone could reprogram fibroblasts into iCMs that exhibit classic iCM molecular features and reprogramming trajectories revealed by our single cell dual-omics. In summary, we elucidated the translatome dynamics of iCM reprogramming and identified Ybx1 as a critical translational barrier to iCM generation, removing of which allowed single factor mediated iCM reprogramming.
Abstract P1022: Intravenously Transplanted Mesenchymal Stem Cells--- A New Endocrine Reservoir For Cardioprotection
Authors: Huang Anan , Liu Yue , Qi Xin , Wei Liping , Chen Shang , Huang Haoyan , Zhang Jun , Li Zongjin
Published: November 2022

Aims:
Intravenous mesenchymal stem cells (MSCs) confer considerable cardiovascular benefits to patients. The lung, but not the heart, is the main trapped site for intravenous MSCs. It remains unclear how intravenous MSCs bridge this spatial gap between the lung and the heart to achieve cardiac repair. To investigate how intravenous MSCs, like an endocrine reservoir, secrete specific miRNA-enriched extracellular vesicles (EVs) gradually and continuously to alleviate doxorubicin (Dox)-induced cardiotoxicity.

Methods and results:
MSCs were isolated from human placenta tissue, and multiple molecular imaging was generated by modifying MSCs-DF or MSCs-Gluc transduced with firefly luciferase (
Fluc
) / enhanced green fluorescence protein (eGFP) or
Gaussia
luciferase (Gluc) lactadherin fusion protein. The Dox-induced cardiotoxicity murine model was constructed and an equal sum of MSCs was infused into mice by single high dose (5 х 10
6
MSCs) or repeated low dose (1 х 10
6
MSCs, 5 times) intravenously. Dox-induced cardiotoxicity, characterized by cardiac atrophy, left ventricular dysfunction, and injured myocardium, was alleviated by low doses of repeated MSCs. These cardioprotective effects were attributed to the suppression of GRP78 triggering stress-induced apoptosis of the endoplasmic reticulum (ER) in cardiomyocytes. Our results confirmed that miR-181a-5p in EVs derived from MSCs (MSC-EVs) inhibited GRP78. Intravenous MSCs-DF were trapped in the lung vasculature, secreted EVs into serum, which was confirmed by the detection of eGFP
+
EVs. Gluc activity increased in serum EVs from mice administrated with MSCs-Gluc. MiR-181a-5p, which inhibits GRP78 with high efficacy, expressed an increase in serum EVs and myocardium after injecting repeated low-dose MSCs. Up-regulation or down-regulation of miR-181a-5p levels in MSC-EVs had enhanced or weakened therapeutic effects on Dox-induced cardiotoxicity by modulating ER stress-induced apoptosis.

Conclusion:
This study identifies intravenous MSCs, recognized as an endocrine reservoir, secrete EVs enriched with miR-181a-5p in the blood to confer indirect cardioprotection against Dox-induced cardiotoxicity.
Abstract P1030: LIN28a-induced Metabolic Reprogramming Regulates New Myocyte Formation In The Heart Via Lncrna-H19
Authors: Rigaud Vagner O , Hoy Robert , Kurian Justin , Zarka Clare , Behanan Michael , Brosious Isabella , Pennise Jennifer , Patel Tej K , Kraus Lindsay , Mohsin Sadia , Houser Steven R , Khan Mohsin
Published: November 2022

Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear and diploid cardiomyocytes (MNDCMs). Upon maturation, MNDCMs become binucleated or polyploid and exit the cell cycle. Interestingly, CM metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative MNDCMs enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism, in cardiac repair following injury. LIN28a was found as primarily active during cardiac development and rapidly decreases after birth. LIN28a reintroduction at P1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing CM cell cycle activity in LIN28a transgenic pups compared to WT littermates. Moreover, LIN28a overexpression extended CM cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated CM apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared to WT littermate controls. Alternatively, LIN28a small molecule inhibitor attenuated pro-reparative effects of LIN28a on the heart. Mechanistically, Neonatal rat ventricular myocytes (NRVMs) overexpressing LIN28a showed increased glycolysis, ATP production and levels of metabolic enzymes compared to control. LIN28a immunoprecipitation followed by RNA sequencing (RIPseq) in CMs isolated from LIN28a injured hearts identified lncRNA-H19 as its most significantly altered target. Ablation of lncRNA-H19 blunted LIN28a-induced enhancement on CM metabolism and cell cycle activity. Collectively, LIN28a reprograms CM metabolism and promotes persistence of MNDCMs in the injured heart enhancing pro-reparative processes thereby linking CM metabolism to regulation of ploidy/nucleation and repair in the heart.
Abstract P1036: Role Of Mitochondrial Ribosomal Protein L7/l12 (mrpl12) In Diabetic Ischemic Heart Disease
Authors: Rai Amit K , Lee Brooke , Sanghvi Shridhar , Tomar Dhanendra , Chandrasekera Dhananjie , Gopala Krishna Sreejit , Ponnalagu Devasena , Khan Mahmood , Singh Harpreet , Nagareddy Prabhakara R , Goukassian David A , Katare Rajesh , Koch Walter J , Kishore Raj , Garikipati Venkata
Published: November 2022

Myocardial infarction (MI) is a significant cause of death in diabetic patients. In addition, growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. The current study aimed to investigate the role of mitochondrial ribosomal protein L7/L12 (MRPL12) in mouse models of type II diabetes (db/db mice)and high-fat diet (HFD) mice with or without induction of MI and human hearts with or without diabetes (n=7) .Data analysis revealed an increase in MRPL12 levels in the LV tissue of HFD fed mice with MI than in LV tissues of low-fat diet-fed mice with MI, whereas MRPL12 levels remained unchanged in the db/db mice with MI. Intriguingly, we found increased MRPL12 levels in atrial appendage tissue of diabetic patients with ischemic heart disease compared to non-diabetic patients. We utilized human cardiomyocyte cell-line (AC-16) as surrogate models to delineate the mechanisms; surprisingly, adenovirus-mediated overexpression of MRPL12 with or without hyperglycemia in AC-16 cardiomyocytes does not affect mitochondrial OXPHOS . In addition, overexpression of MRPL12 had no effect on the mitochondrial ROS, mitochondrial membrane depolarization, and caspase activity in AC-16 cardiomyocytes. Whereas RNA interference (RNAi)-mediated MRPL12 silencing remarkedly reduced mitochondrial oxidative phosphorylation in AC-16 cells without any stress. In addition, knockdown of MRPL12 increased mitochondrial membrane depolarization mitochondrial ROS and reduced maximal respiratory capacity of mitochondria without any stress. Overall, our results provide new insights into the role of MRPL12 in the pathophysiology of MI in diabetes.
Abstract P1037: Polycythemia Vera Is Not Associated With Higher Inpatient Mortality In Patients Hospitalized For Myocardial Infarction: A Nationwide Analysis
Authors: Orji Richard , Ukenenye Emmanuel , Samuel Shirly , Khabsa Mariam , Yassa Gizem , Waraich Saad , shayuk Maryna
Published: November 2022

Introduction:
Polycythemia vera (PV) is a well-known myeloproliferative disorder with an abundance of red blood cells as well as white blood cells and platelets. This condition is also known as hyperviscosity syndrome (HVS). Myocardial infarction (MI), a lethal disease, occurs due to impaired coronary blood supply to the heart. The hyperviscosity of coronary arterial blood flow in PV patients is associated with an increased risk for coronary syndrome. However, our study sought to determine if PV worsens the outcomes or aggravates ongoing MI in hospitalized patients.

Methods:
We queried the National Inpatient Sample (NIS) database from 2016 to 2019. The NIS is the largest inpatient hospitalization database in the United States. The NIS was searched for hospitalization of adult patients with MI as a principal diagnosis with and without PV as a secondary diagnosis using ICD-10 codes. The primary outcome was inpatient mortality. The secondary results were Acute kidney injury (AKI), Cardiogenic shock (CS), Cardiac arrest (CA), Total hospital charge (THC), and length of stay (LOS). Multivariate logistic and linear regression analysis were used accordingly to adjust for confounders.

Results:
About 2,622,939 patients were admitted for MI; 0.091% (2,385) had underlying PV. Cohorts with PV vs No PV had a mean age of 69.2 years [CI 68.0 - 70.5] vs 66.9 years [CI 66.9 - 67.0]; male (66.2% vs 62.3%), female (33.8% vs 37.7%); white (85.3% vs 73.4%), black (5.6% vs 11.4%), and Hispanic (5.0% vs 8.8%). Compared to patients without PV, patients admitted with coexisting PV had similar inpatient mortality (5.7% vs 4.6%, AOR 1.17, 95% CI 0.79 - 1.73, P 0.437), AKI (20.3% vs 19.9%, P 0.546), CS (8.0% vs 6.3% P 0.222), CA (2.5% vs 2.9% P 0.690), THC (IRR 1.05, 95% CI 0.95 - 1.17, P 0.342), and LOS (IRR 1.06, 95% CI 0.95 - 1.18, P 0.277).

Conclusion:
Patients admitted primarily for MI with co-existing polycythemia vera had similar inpatient mortality. There was no difference in AKI, CS, CA, THC, and LOS in both groups. Phlebotomy in hospitalized Patients with MI and co-existing PV should be carried out with caution due to the risk of hypovolemia and possibly exacerbating the infarct. Additional studies are required to elaborate on the delicate management of this patient group.
Abstract P1039: Overexpressing Mitochondrial Transcription Factor A Protects Against Myocardial Stunning And Improves Mortality In A Mouse Model Of Cardiac Arrest
Authors: Rutledge Cody , Redding Kevin , Dezfulian Cameron , Kaufman Brett
Published: November 2022

Background:
Cardiac arrest (CA) affects over 600,000 Americans every year and is associated with overwhelming mortality. Patients who survive CA frequently develop multi-system injury, including cardiac dysfunction and reduced cardiac ejection fraction (EF). Nearly 25% of these patients die from cardiogenic shock. This cardiac injury is primarily attributed to ischemia-reperfusion injury. Mitochondria make up 30% of the heart by volume and are a major site of ischemia-reperfusion damage. Such damage affects mitochondrial DNA (mtDNA), but these changes may be limited by manipulating mitochondrial transcription factor A (TFAM). We investigated the role of cardiac-specific TFAM overexpression on cardiac function after CA in a mouse model.

Methods:
Wild-type (WT) and transgenic mice featuring cardiac-specific TFAM overexpression (TFAM OE) were subjected to 8-min of asystole by direct injection with potassium chloride, followed by cardiopulmonary resuscitation. Surviving mice were assessed by echocardiography at 1-day, 1-week, and 4-weeks and monitored for survival over the time frame. Hearts were collected and sent for bulk RNA sequencing as well as assessment of mtDNA copy number, mitochondrial morphology, and protein expression.

Results:
TFAM OE mice express 40% more TFAM than WT littermates. TFAM OE mice had protected cardiac EF (51.11±2.95%) compared to WT mice (38.49±3.76%) at one day and improved survival over 4 weeks. Bulk-RNA sequencing implicated changes to ribosomal expression and protein synthesis in the arrest TFAM OE mice. Western blot analysis of 1-day post-arrest hearts demonstrated increased expression of multiple heat shock proteins and increased phosphorylation of eukaryotic translation initiation factor 4E-binding protein (4E-BP1), suggesting that TFAM OE mice support protein translation to aid in cardiac recovery after arrest.

Conclusions:
Cardiac TFAM overexpression protects cardiac function and survival after cardiac arrest. Early evidence suggests that this protection may be mediated by supporting protein translation, a novel potential pathway for improving outcomes after arrest.
Abstract P1042: Endothelial Hif-2α Expression Prevents Against Myocardial Infarction Induced Heart Failure And Cardiac Microvascular Barrier Dysfunction
Authors: Ullah Karim , Ai lizhuo , Liu Lifeng , Zhang Qin , Pan Kaichao , Sitikov Albert , Liao James K , Wu Rongxue
Published: November 2022

Background:
HIF pathway is quickly activated during myocardial ischemia after myocardial infarction(MI), and cardiac microvascular leakage contributes to heart tissue damage. HIF2α isprofoundly expressed in cardiac endothelial cells (ECs) and the embryonic deletion of HIF2Aresults in increased vascular permeability and aberrant ECs behavior. However, the direct roleof endothelial cell-specific HIF2α (ecHIF2α) in ischemic heart disease is not known. Wehypothesized that ecHIF2α expression in response to myocardial infarction (MI) is protectiveagainst heart failure through the reduction of cardiac ECs apoptosis and inflammation.

Methods and Results:
To address our hypothesis, we generated EC-specific inducible-HIF2α knockout mice (ecHIF2α
-/-
) by crossing

Hif2a
flox/flox
mice with

Cre ERT2 mice. To assess the functional role of HIF2α inischemic heart injury, we ligated the proximal left anterior descending coronary artery to induceMI using the same age and gender-matched
ecHIF2α

-/-

and control (
Hif2a

flox/flox

) mice. Cardiacfunction was determined by echocardiography after two and four weeks of ligation. Analysis ofechocardiography revealed worsened heart function, and Masson’s Trichrome stain displayedincreased fibrosis in
ecHIF2α

-/-

mice.
In vitro
, ECIS analysis of isolated cardiac microvascularendothelial cells showed decreased endothelial barrier function in
ecHIF2α

-/-

cells. In addition,hypoxic stimulation reduces the tube formation capacity in ecHIF2α-/- cells and is sensitive tohypoxia-induced early-stage apoptosis. Deletion of HIF2α, as well as its binding partner ARNT,increased the expression of several inflammatory genes, including IL-6. Interestingly,overexpression of ARNT alone abolishes the HIF2α deletion-induced inflammatory geneexpression. IL-6 protein levels in HIF2α deleted human aortic endothelial cells (HAoEC) show asignificant reduction (n=3-5, p<0.001) in ARNT overexpressed ECs.

Conclusion:
Collectively our data revealed an essential role of endothelial HIF2α/ARNT in maintaining cardiacfunctions by increasing endothelial barrier function and decreasing inflammation. Therefore,HIF2A/ARNT could provide a potential therapeutic target for the treatment of ischemic heartdisease.
Abstract P1045: Targeting The EF-hand Containing Domain 1 Protein (EFHD1) In Cardiac Disease
Authors: Eberhardt David R , Yin Xue , Balynas Anthony , Lee Sandra , Walsh Maureen , Andrea Corbin , Funai Katsuhiko , Sachse Frank , Chaudhuri Dipayan
Published: November 2022

Mitochondrial dysfunction due to calcium overload is common in heart disease and results in heart failure. In a mouse model of mitochondrial cardiomyopathies we noted a substantial downregulation of the gene encoding for the EF hand containing domain 1 protein (EFHD1). EFHD1 is a mitochondrial protein which binds calcium and has been shown to stimulate both apoptosis and mitoflashes. These processes involve mitochondrial calcium overload. We hypothesized that EFHD1 down regulation could represent a natural response by failing hearts to prevent mitochondrial calcium overload by increasing the calcium handling of mitochondria. We therefore studied the calcium handling properties of mice where EFHD1 had been knocked out using CRISPR Cas9. These mice are viable and we have found no adverse cardiac or metabolic phenotypes. We find that EFHD1 is expressed in the intermembrane space of the mitochondria and associates strongly with endoplasmic reticulum (ER)-associated mitochondrial membranes. Mitochondria isolated from Efhd1 knockout mice also exhibited a <250±10 % improvement in calcium retention in liver and neonatal mouse hearts were 3 times more resistant to hypoxia. Further, Efhd1-/- cardiomyocytes exhibit reduced ROS and mitoflash events at both baseline and following ischemia-reperfusion. These findings suggest that EFHD1 participates in ER-mitochondrial calcium exchange and that EFHD1 ablation is cardioprotective during ischemia-reperfusion.
Abstract P1046: An Oxygen Gradient Microphysiological System To Model The Myocardial Infarct Border Zone
Authors: Rexius-Hall Megan L , Khalil Natalie N , Escopete Sean S , Li Xin , Hu Jiayi , Yuan Hongyan , Parker Sarah , McCain Megan L
Published: November 2022

After a myocardial infarction, the border zone is characterized by an oxygen (O
2
) gradient located at the interface between the injured, hypoxic cardiac tissue and adjacent normoxic, viable tissue. Yet, the impact of an O
2
gradient on cardiac function is not well understood because there is a lack of in vitro experimental platforms for generating such biomimetic O
2
landscapes. This unmet need has prevented systematic investigation into how hypoxic-normoxic intercellular communication affects cardiac function. In this study, a microphysiological system was engineered to generate and expose engineered cardiac tissues to a stable O
2
gradient as a “Border-Zone-on-a-Chip” model. Briefly, a microfluidic gas supply channel network was overlaid with a thin, gas-permeable polydimethylsiloxane membrane on which engineered cardiac tissue composed of neonatal rat ventricular myocytes was cultured. Constant perfusion of compressed gases through separate microfluidic channels and rapid diffusion of the gas conditions across the thin membrane produced an O
2
gradient. Functional and transcriptomic analysis revealed biological responses were uniquely regulated by an O
2
gradient when compared to engineered tissues exposed to global, homogeneous O
2
levels. In the gradient, there was a significant increase in the calcium transient time to peak (85%, p=0.0007) and tau time constant of decay (49%, p=0.006). There was a significant decrease in the longitudinal calcium wave propagation velocity (35%, p=0.03), diastolic stress (47%, p=0.01), and peak systolic stress (47%, p=0.01). Overall, the gradient exhibited hallmarks of arrhythmogenesis, hypocontractility, and activation of inflammation after 4 h of O
2
modulation. Importantly, these changes were distinct from those observed in tissues exposed to uniform normoxia or hypoxia, demonstrating distinct regulation of cardiac tissue phenotypes by an O
2
gradient. Studies are ongoing to elucidate a possible paracrine mechanism involved in the observed effects. By gaining functional and mechanistic insight into intercellular communication activated in O
2
gradients, microphysiological systems can direct future therapeutic strategies aimed at minimizing or preventing cardiac hypoxic injury.
Abstract P1047: Imatinib Attenuates Reperfusion Injury In A Rat Model Of Acute Myocardial Infarction
Authors: Konijnenberg Lara S , Uthman Laween , Veltien Andor , Kuster Carolien T , Luiken Tom T , De Waard Guus A , Lemmens-Hermans Kitty , de Haas-Cremers Karin , Kea-te Lindert Mariska , Daviran Deniz , Thijssen Dick H , Akiva Anat , Nijveldt Robin , van Royen Niels
Published: November 2022

Background:
Reperfusion of an occluded coronary artery is often accompanied by injury of the microvasculature (MVI), which portends a worse long term prognosis after an acute myocardial infarction (AMI). In previous experimental studies, the tyrosine-kinase inhibitor imatinib has shown to reduce vascular leakage and therefore could have a potential role to prevent MVI. This study aims to provide a potential therapeutic approach using imatinib to attenuate reperfusion injury.

Methods:
First, 16 isolated male Wistar rat hearts were randomly assigned to 10 μM imatinib or placebo and subjected to 40 min of global ischemia followed by 120 min of reperfusion ex vivo in a Langendorff setup. Global infarct size and mechanical function were assessed. Second, 25 male Wistar rats were randomly assigned to 30 mg/kg imatinib or placebo intravenously and subjected to 45 min of left anterior descending coronary artery ligation followed by 180 min of reperfusion in vivo. Cardiovascular magnetic resonance imaging was performed to assess infarct size and cardiac function. Subsequently, hearts were perfused ex vivo with a fluorescent vascular leakage tracer and used for fluorescence- and electron microscopy.

Results:
In isolated hearts, imatinib reduced global infarct size (imatinib 36.2±7.9 vs placebo 50.0±8.0% infarcted area/total area, p<0.01), improved end diastolic pressure (imatinib 44.2±8.0 vs placebo 59.5±13.5 mmHg, p<0.05) and rate pressure product recovery (imatinib 39.0±6.8 vs placebo 26.2±7.6% of baseline, p<0.05). In vivo, imatinib reduced infarct size (imatinib 12.6±8.8 vs placebo 25.6±9.3% infarcted area/left ventricle, p<0.01), but did not improve global cardiac function. At the end of reperfusion, imatinib showed lower vascular resistance, higher coronary flow and less microvascular leakage (all p<0.05). At the ultrastructural level, imatinib showed better preserved microvascular integrity compared to placebo.

Conclusion:
This study provides evidence that imatinib attenuates reperfusion injury in an ex vivo and in vivo AMI rat model, mainly by protection of the coronary microcirculation. These data warrant future work to examine the potential of imatinib to reduce reperfusion injury in patients with AMI.
Abstract P1048: Dual Myristic Acid And Trans Activator Of Transcription Conjugated Peptide Facilitates Delivery Of Protein Kinase C Beta II Peptide Inhibitor To Attenuate Myocardial Ischemia/Reperfusion Injury
Authors: Clair Logan , Ramsarran James , Augustin Taurai , Stinson Cameron , Dean Tameka , CHEN Qian , Barsotti Robert , Young Lindon
Published: November 2022

Peptides conjugated to myristic acid (myr) or trans activator of transcription (tat) have long been used to increase cell permeability via simple diffusion and endocytosis, respectively. Previously myr-PKCβII inhibitor (myr-PKCβII-) exerted cardioprotective effects in myocardial ischemia/reperfusion (I/R) and inhibited superoxide (SO) release from rat leukocytes by 40% (20 μM). By contrast, dual conjugated myr-tat-PKCβII inhibitor (myr-tat-PKCβII-) inhibited SO release by 90% at the same concentration. The purpose of this study was to determine the potency of myr-tat-PKCβII- compared to myr-PKCβII- in mitigating infarct size and restoring cardiac function in an ex-vivo rat model of myocardial I/R injury.

Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to global I(30-min)/R(50-min) . Left ventricular cardiac function was recorded using a pressure transducer. Treatments were infused during the first 5 min of R. After R, the hearts were sectioned (2 mm) from base to apex and stained with 1% triphenyltetrazolium chloride. Infarcted tissue was excised to determine infarct size (i.e. infarct tissue weight/ total tissue weight). Data were analyzed using ANOVA Fisher’s LSD analysis.

Compared to untreated controls (23.2±3.1%, n=17), myr-tat-PKCβII- exerted a significant and similar decrease in infarct size that was observed from 100 nM (9.7±0.59%, n=3, p<0.05), 1 nM (10.0±2.3%, n=5, p<0.05), to 100 pM (12.6±2.3%, n=5, p<0.05); but not at 10 nM (13.8±3.6%, n=5). Cardiac function for all treatment groups did not significantly improve from control. However, at 100pM (1070±170 mmHg/s), +dP/dt max was significantly improved compared to all other treatment groups (p<0.05).

The addition of myr to tat-conjugated peptides improved the cardioprotective effects ~200-fold compared to myr-PKCβII- and ~2000-fold compared to native peptide presumably by enhanced intracellular delivery of cargo. The ~50% reduction in infarct size suggests that myr-tat-PKCβII- is cardioprotective in I/R. Future studies will test our most effective dose determined in ex vivo hearts to restore post-reperfusion cardiac function combined with significant cardiac salvaging effect (reduced infarct size) in a porcine myocardial I/R model.
Abstract P1058: Whole-cell Mechanical Loading And Unloading Triggers More Post-translational Modifications In α-actinin Than Myosin Activators And Inhibitors
Authors: Solis Christopher , DiNello Elisabeth , Warren Chad M , Dittloff Kyle , Solaro R John , Russell Brenda
Published: November 2022

The hypothesis tested is that unloading of mechanical forces affects acetylation, ubiquitination, and phosphorylation of the actin-binding protein α-actinin located in the Z-disc. We applied targeted proteomics to interrogate the post-translational modification changes during loading and unloading. Cell morphology and post-translational modifications were determined in a mechanical intervention consisting of 1 Hz cyclic strain for 24 hr (loaded) followed by 6 hr rest (unloaded) of cultured neonatal rat ventricular myocytes (NRVMs). This was compared to a chemical intervention consisting of treating NRVMs with the myosin inhibitor Mavacamten (1 μM, 6hr) or activator Omecamtiv Mecarbil (0.5 μM, 6hr). Quantitative immunofluorescence showed both chemical and mechanical loading increased α-actinin content while Mavacamten decreased the α-actinin content. Mass spectrometry analysis of affinity-purified α-actinin revealed that the mechanical intervention led to increased levels of post-translational modifications compared with the chemical interventions. Specifically, α-actinin ubiquitination increased with mechanical loading-unloading; acetylation decreased with mechanical loading-unloading and increased with mechanical loading; and phosphorylation remained unchanged with mechanical loading-unloading but increased with mechanical loading. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that Mavacamten increased the dynamics of overexpressed YFP-tagged α-actinin and a GFP-tagged CapZ in NRVMs when compared to Omecamtiv Mecarbil treatments and controls. Overall, the results suggest a link between sarcomere homeostasis and mechanical forces via mechanisms involving acetylation, phosphorylation and ubiquitination of α-actinin and a second Z-disc protein, CapZ. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis. Funded by NIH grants HL151825 (CS) and HL62426 (RJS, BR, and CMW).
Abstract P1060: Sarcomeric Protein Phosphorylation Protects Mouse Hearts From Ischemia/reperfusion Injury
Authors: Wang Buheng , Jun Seungho , Keceli Gizem , Rostampour Sara , Paolocci Nazareno , Li Yuejin
Published: November 2022

Introduction:
Post-translational modifications of cardiac troponin I (cTnI), an essential sarcomeric protein, have been known to play a vital role in regulating heart function. Site-specific phosphorylation on cTnI Ser199 is upregulated in human ischemic heart failure, negatively affects diastolic function but preserves ejection fraction in mice
in vivo
, and promotes contractility recovery after ischemia/reperfusion (I/R) in male isolated mouse hearts. However, its cardioprotective mechanism is largely unknown.

Hypothesis:
We assessed the hypothesis that cTnI Ser199 hyperphosphorylation protects hearts during I/R via down-regulating reactive oxygen species (ROS).

Methods:
Due to the current methodology limitation, there is no way to phosphorylate one specific site on cTnI
in vivo
. We, therefore, generated a transgenic mouse model named TgD that carried Serine (S) to Aspartic Acid (D) mutation at cTnI Ser200 (equivalent to Ser199 in human sequence) to mimic the site-specific hyperphosphorylation, which was a widely accepted method for
in vivo
studies on sarcomeric protein phosphorylation. The cardiac function was examined using Langendorff isolated hearts with pacing at baseline and after 30-minute global ischemia followed by 2-hour reperfusion.

Results:
At baseline, TgD hearts (both genders, 7-8 mice/gender, age 3-5 months) showed comparable rate pressure product (RPP), left ventricular developed pressure (LVDevP), and d
P
/d
t
max
but decreased d
P
/d
t
min
relative to wildtype (WT) littermates. After I/R, TgD presented significantly better RPP, LVDevP, d
P
/d
t
max
, and d
P
/d
t
min
with remarkable contractile function recovery rate compared to WT (i.e., 47% ±2% vs. 24%±2% in RPP, P<0.05) in both genders. The level of total ROS was similar in TgD and WT hearts at baseline. Although the ROS increased after I/R, it remained at a significantly lower level in post-I/R TgD hearts than WT controls, associated with differentially altered activities of antioxidant enzymes.

Conclusions:
In conclusion, human heart-failure-related cTnI Ser199 hyperphosphorylation protects heart function during global I/R in isolated mouse hearts in both genders
.
The protective mechanism, at least in part, involves the downregulation of ROS.
Abstract P1061: Crosstalk Between Acetylation And Methylglyoxal Modifications On Myofilament Proteins Affects Sarcomere Function
Authors: Delligatti Christine , Papadaki Maria , Martin Thomas , Kirk Jonathan A
Published: November 2022

Diabetes doubles the risk of developing heart failure, independent of coronary artery disease and hypertension. The mechanisms connecting these two diseases are poorly understood, although we have previously shown one connection is likely due to methylglyoxal (MGO), a highly reactive glycolysis byproduct that can irreversibly modify lysine (K) and arginine (R) residues in a process called glycation. We found MGO glycation of the myofilament decreased calcium sensitivity and maximum calcium activated force in diabetes. Here we hypothesized MGO may further impact function by competing for lysine residues that would otherwise be targets for acetylation, which has been shown to impact cardiac function. To assess this hypothesis, non-failing human left ventricular (LV) myocardial tissue was exposed to MGO (100 μM) or vehicle and then the treated with acetic anhydride in acetonitrile (300 μM, Ac
2
O-ACN) overnight. Ac
2
O-ACN increased myofilament protein acetylation in both groups, but this effect was greatly reduced in samples pre-treated with MGO, suggesting these two PTMs do compete for a subset of lysine residues. To discover the specific sites of this acetylation-glycation crosstalk, we compared mass spectrometry data sets from diabetic humans, MGO treated mouse myocytes, and acetylated myocytes, which revealed sites of crosstalk on actin, myosin, and myosin light chains - proteins essential for normal contractile function. Importantly, these glycation-acetylation crosstalk sites were found in human samples and the data suggest the balance of these may change in disease states. To explore the functional consequence(s) of this crosstalk, we incubated human LV isolated skinned myocytes with Ac
2
O-ACN (300 μM, overnight), which enhanced function by significantly increasing calcium sensitivity. However, pre-treatment with MGO (100 μM) completely blocked this functional effect of acetylation. Together, these data demonstrate that glycation and acetylation compete for certain myofilament lysine residues, and that glycation can block the beneficial functional effects of acetylation. These results provide insight into the molecular mechanisms behind diabetic cardiomyopathy and the utility of targeting acetylation clinically.
Abstract P1062: Regional Differences In Sarcomeric Glycogen Synthase Kinase 3β Maintain Normal Chamber Level Function
Authors: Stachowski-Doll Marisa , Door Michaela , Kirk Jonathan A
Published: November 2022

The structure of the left ventricle is spatially and temporally heterogeneous. The heart’s electrical system helps synchronize these events, but mechanical events play an important role as well. However, experimental evidence of the regional mechanical processes that underpin a “normal contraction” remain largely unknown. Despite the differences in deformation between the anterior and posterior walls, sarcomere lengths during filling and ejection are the same in these regions. One explanation for this disconnect is the myocyte’s force production is different despite similar sarcomere lengths, a relationship known as Length Dependent Activation (LDA). Indeed, a modeling study suggested LDA is a critical regulator of mechanical synchrony in the left ventricle. We recently found the serine/threonine kinase GSK-3β localizes to the z-disc and can modulate LDA, and thus hypothesized it may play a role in maintaining normal cardiac synchrony. We used inducible cardiomyocyte specific GSK-3β KO mice and GSK-3βfl/fl/Cre- littermate Con treated with tamoxifen in which GSK-3β was KO for three weeks. We assessed regional differences in GSK-3β protein levels and found that Con mice had substantial regional heterogeneity: 50% more cytosolic GSK-3β in the posterior wall compared to the anterior wall; however, the myofilament-bound GSK-3β was the opposite, with 50%
less
GSK-3β in the posterior wall. This regional heterogeneity was lost in KO mice, in which protein levels were homogenously low. There were no differences detected by m-mode echo between Control and KO mice. However, strain analysis revealed a significant interaction (p=0.037) between genotype and region (Anterior vs. Posterior), in which regional differences in Systolic Strain Rate were ablated in the knockout. These data suggest that the regional differences in GSK-3β sarcomeric levels were important for maintaining regional functional heterogeneity. We further observed that sarcomeric GSK-3β levels (but not cytosolic) decreased in a mouse myocardial infarction model, which these findings suggest could worsen mechanical dyssynchrony post-infarct. Perhaps a possible therapy for mechanical dyssynchrony could involve regional restoration of sarcomeric GSK-3β levels to restore synchrony.
Abstract P1065: Deletion Of Arginine 14 Increases Phospholamban Oligomerization And Disrupts The Cardiac Force-Frequency Relationship
Authors: Cleary Sean R , Phillips Taylor A , Cho Ellen E , Robia Seth L
Published: November 2022

The Bowditch effect is a physiological phenomenon where accelerated cardiac pacing leads to an increase in the contractile force of the heart. While this has largely been attributed to a frequency-dependent increase in L-type calcium channel influx, evidence suggests intracellular calcium handling proteins must also play an active role. Knock-out of phospholamban (PLB), the inhibitor of the Ca ATPase (SERCA), virtually abolishes the Bowditch effect in mice, suggesting PLB is a major determinant of the cardiac force-frequency relationship. However, no molecular mechanism has explained this frequency-dependent function of PLB. Our lab has previously shown that SERCA loses affinity for PLB when intracellular calcium is elevated. Here, FRET microscopy revealed dynamic decreases in PLB-SERCA binding in response to intracellular calcium elevations. PLB monomers freed from SERCA were rapidly incorporated into PLB pentamers. Interestingly, unbinding of PLB from pentamers was very slow and delayed the recovery of PLB-SERCA complexes by several seconds after calcium elevations. In a computational model of these regulatory binding dynamics, PLB pentamers sequestered inhibitory monomers away from SERCA at fast heart rates. By lowering PLB inhibition of SERCA with increasing cardiac pacing, PLB pentamers serve as a major contributor to the Bowditch effect. Notably, deletion of arginine 14 of PLB causes arrhythmogenic cardiomyopathy and has been thought to destabilize PLB pentamers. Surprisingly, we found R14del-PLB enhanced pentamer stability and slowed the rate of PLB exchange from pentamers to SERCA. We hypothesize that R14del mutation may disrupt frequency-dependent changes in SERCA regulation by PLB during exercise.