bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1105: Dissecting The Roles Of Vegfba And Pgfb In Zebrafish Cardiac Regeneration
Authors: Wang Zhenyu , Stainier Didier
Published: October 2023

Vascular endothelial growth factor (VEGF) family members promote angiogenesis, regulate inflammation, antagonize oxidative stress, and regulate lipid metabolism, which present potential therapeutic value for coronary heart disease. However, all of their roles during cardiac regeneration are still largely unknown. Unlike mammals, zebrafish are able to fully regenerate their heart after injury. In zebrafish, Vegf factors are regulated in different manners after cardiac cryoinjury, indicating functional diversity during cardiac regeneration. Here, we focus on
vegfba
and placental growth factor b (
pgfb
), which have been much less investigated in regenerative contexts, and were found to be persistently down-regulated in injured hearts before 5 days post cryoinjury (dpci) according to our previous dataset. We generated HOTcre transgenic lines to control the overexpression of
vegf
genes in a spatially and temporally restricted fashion during cardiac regeneration. Global overexpression of
vegfba
had no significant effect on coronary vessels, whereas it promoted the proliferation of cardiomyocytes at 7 dpci. In addition, global overexpression of
pgfb
significantly increased the vessel coverage in the wound at both 96 hpci and 7 dpci. These results suggest their possible roles in promoting myocardial regeneration and revascularization. However, myocardium-specific overexpression of
vegfba
and
pgfb
significantly reduced coronary vessel coverage within the injured area and decreased the proliferation and dedifferentiation of cardiomyocytes, indicating possible disruption of revascularization and regeneration caused by their misexpression. To further study the mechanism underlying these phenotypic differences, we are performing transcriptomic analysis to identify the differentially regulated genes and related signaling pathways. This project will provide new candidate cues that may help understand the mechanisms of coronary revascularization and cardiac regeneration in zebrafish, with potential therapeutic implications of achieving better directed and enhanced regeneration upon coronary heart disease.
Abstract P1116: Histone Deacetylase Complexes Regulate Direct Cardiac Conversion From Human Fibroblasts
Authors: Wang Meimei , Lu Yu-An , Zhou Xinyue , Lu Rui , Zhou Yang
Published: October 2023

Direct cardiac reprogramming is a promising approach to generate functional cardiomyocytes from non-myocytes for regenerative medicine, drug screening, and disease modeling. This method involves the transformation of specialized cells into functional cardiomyocytes using specific transcription factors or molecular signals. Epigenetic modulators, such as histone deacetylase (HDAC) complexes, may play a critical role in chromatin remodeling underlying cardiac reprogramming.

To investigate the role of HDAC complexes in direct cardiac conversion, induced cardiomyocyte-like cells (iCMs) were derived from human fibroblasts through ectopic expression of reprogramming factors, and the changes of histone acetylation were evaluated by western blotting. The results showed decreased levels of H3K9ac, H3K14ac, and H3K27ac along with direct cardiac reprogramming, suggesting the significant chromatin remodeling and gene regulation.

A loss-of-function screen of all the components associated with HDAC complex was performed to determine the responsible histone acetylation modifiers. The screen results showed that most of the HDAC components are necessary for direct cardiac reprogramming. Further investigation focused on a newly identified HDAC complex, mitotic deacetylase complex (MiDAC), consisting of mitotic deacetylase associated SANT domain protein MiDEAS (ELMSAN1), DNTTIP1, and HDAC1. Knocking down each of the MiDAC components led to similar reprogramming repression and cell death, indicating that MiDAC plays a critical role in cardiac reprogramming. In addition, the expression analysis of DNTTIP1 and HDAC1 showed a gradual decrease throughout the conversion, while ELMSAN1 diminished at the early stage of reprogramming, suggesting that ELMSAN1 is likely required for MiDAC stability. Furthermore, overexpression of ELMSAN1 significantly improved reprogramming efficiency and led to the increase of H3K9ac, H3K14ac, and H3K27ac levels globally, suggesting that ELMSAN1 and MiDAC might be responsible for the regulation of such histone modifications during the process.

Overall, our findings provide novel insights into the epigenetic regulation of direct cardiac reprogramming and reveal the potential role of MiDAC in this process.
Abstract P1118: Engineering A Patient-specific Heart On A Chip Model Of Hypoplastic Left Heart Syndrome
Authors: Verma Riya , Rexius-Hall Megan L , McCain Megan L , Subramanyan Ram Kumar
Published: October 2023

Hypoplastic Left Heart Syndrome (HLHS) is the most expensive congenital cardiac disease and is characterized by underdevelopment of the left side of the heart. The surgical intervention leads to the developmentally disadvantaged right ventricle performing systemic circulation. As such, a subset of HLHS patients develops progressive heart failure that results in limited transplant-free survival. There are no animal models to study the molecular and functional properties of HLHS cardiomyocytes, limiting our understanding of disease mechanisms and human genotype-phenotype relationships. To address this, we are creating a physiologically and functionally relevant ”heart on a chip” model of HLHS hiPSC-derived cardiomyocytes (CMs). Induced Pluripotent Stem Cells (iPSCs) were derived from cord blood mononuclear cells isolated from the umbilical cord blood from 8 antenatally diagnosed HLHS children at birth. Control (iPSCs) from 3 healthy subjects were derived from peripheral blood mononuclear cells. We then differentiated ventricular CMs from these iPSCs using StemDiff ventricular CM kit. We observed HLHS iPSCs formed ~50% fewer CMs compared to healthy controls. We studied calcium handling by iPSC-CMs with Fluo-4 fluorescent calcium dye in HLHS and Healthy iPSC-CMs. We observed that calcium transients in HLHS-CMs have half the beat rate, 1.4 times longer rise time to rise, 1.5 times higher time constant (Tau), compared to Healthy CMs, suggesting that HLHS CMs have dysfunctional calcium handling compared to healthy CMs. Our results demonstrate that in an in vitro model of iPSC-derived CM, HLHS patient CMs demonstrate reduced formation and defective calcium handling, which may potentially explain the pathogenesis of heart failure in HLHS patients. In ongoing experiments, we are evaluating diastolic and systolic stress generation in these cells. We will also establish the underlying molecular mechanisms of functional defects in HLHS CMs and screen potential drug therapies for single-ventricle patients.
Abstract P1120: Chromobox 7 Represses Cardiomyocyte Proliferation, And Its Genetic Ablation Promotes Cardiac Regeneration Following Myocardial Injury
Authors: Cho Kyuwon , Andrade Mark , Bae Seongho , Kim Sangsung , Kim Jin Eyun , Jang Er Yearn , Lee Sangho , Husain Ahsan , Sutliff Roy L , Calvert John W , Park Changwon , Yoon Young-sup
Published: October 2023

Background:
Cardiomyocyte (CM) proliferation decreases after birth but the underlying mechanism is poorly understood. Chromobox 7 (CBX7) regulates the cell cycle but its role in CM proliferation is unknown.

Methods:
We profiled CBX7 expression in the mouse hearts via qRT-PCR, western blotting, and immunohistochemistry. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-cre;Cbx7
fl/+
and Myh6-MCM;Cbx7
fl/fl
, respectively). We then measured CM proliferation by immunostaining. To examine the role of CBX7 in cardiac regeneration, we employed neonatal cardiac apical resection and adult myocardial infarction (MI) models. We examined the mechanism of CBX7-mediated repression of CM proliferation via co-immunoprecipitation, mass spectrometry, and other molecular techniques.

Results:
The mRNA expression of Cbx7 was increased at the perinatal stage and sustained in the postnatal heart. The haplodeficiency of
Cbx7
(Tnnt2-Cre;Cbx7
fl/+
) promoted proliferation of neonatal CMs
in vivo
, leading to increased myocardial wall thickness. Genetic deletion of
Cbx7
in CMs (
Cbx7
iCKO, Myh6-MCM;Cbx7
fl/fl
) at P0 resulted in increased CM proliferation (% of Ki67
+
CMs: vehicle, 9.5 vs. tamoxifen, 18.3, P < 0.01). In response to cardiac apical resection surgery at P7,
Cbx7
iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 44.1 vs. tamoxifen, 54.7, P < 0.05) and less fibrosis at P28 compared to the control. Following the MI surgery, adult (5-months-old)
Cbx7
iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 41.4 vs. tamoxifen, 51.5, P < 0.05) and less fibrosis at one month post-MI compared to the control. Mechanistically, CBX7 interacted with TARDBP and positively regulated its downstream target, RBM38, an inducer of the cell cycle arrest, in a TARDBP-dependent manner. Rbm38 was perinatally upregulated in the mouse hearts and overexpression of Rbm38 reduced proliferation of neonatal CMs.

Conclusions:
CBX7 expression is perinatally increased and inhibits proliferation of CMs by controlling TARDBP/Rbm38 pathway. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult hearts. This is the first study to uncover the role of CBX7 in regulation of CM proliferation and cardiac regeneration.
Abstract P1135: Gene Expression Analysis Of Glycemic Memory In Heart Failure
Authors: Bakshi Sayan , Wende Adam R
Published: October 2023

Diabetes is a growing epidemic, with 37.3 million affected in the US. Diabetic patients are twice as likely to develop cardiovascular disease (CVD). Risk of CVD persists for years even following restoration of glycemic control in type-2-diabetic patients. These findings led to the hypothesis of glycemic memory, i.e., prior changes in glucose levels and delivery may lead to long-term molecular changes predisposing patients to CVD.

We proposed that prior enhanced cellular glucose uptake leads to transcriptional changes that contribute to glycemic memory and enhances susceptibility to heart failure. We used a mouse model of doxycycline (DOX)-inducible cardiomyocyte-restricted glucose transporter 4 (GLUT4; mG4H), where mG4H is overexpressed for 2 wk (ON) and then returned to basal levels (OFF). Harvested left ventricular tissues from 4 groups of male mice (n=3) - ON and matched control (Con), OFF and DOX-matched Con (DCon) - were subjected to RNA sequencing analysis (Adjusted
P
<0.1, Fold-Change>
Abstract P1139: Remodeling Chromatin Structure To Induce And Reverse Heart Failure
Authors: Rosa-Garrido Manuel , Bridges Justin , Chapski Douglas
Published: October 2023

Heart failure is a critical health issue that causes one in nine deaths in the United States. Despite extensive research on gene expression dynamics in the failing myocardium, the mechanisms by which the genome reorganizes itself to reconfigure transcription are poorly understood. CTCF is a crucial protein in chromatin architecture that mediates chromatin looping, acts as an enhancer insulator, and can function as either an activator or repressor of transcription. Our previous research indicates that CTCF plays a crucial role in heart failure, as cardiac-specific knockout of this protein promotes genome organization defects that result in cardiac pathology marked by reduced ejection fraction. To determine whether restoring CTCF levels could reverse this phenotype, we designed an AAV9 vector in which the cardiomyocyte-specific Troponin T promoter drives the expression of CTCF and GFP. Our results show that reintroduction of CTCF into failing CTCF-depleted mouse hearts successfully rescues the pathological phenotype of the animals, characterized by sustained improvement in ejection fraction and left ventricular chamber size in both systole and diastole. Furthermore, RNA-seq experiments reveal numerous differentially expressed genes with opposite transcriptional behavior depending on CTCF levels, including genes involved in chromatin organization (e.g., CHmgb2, Brca1, Smyd2), development of hypertrophy (e.g., Mef2a, Gata4, Mybpc3), extracellular matrix organization (e.g., Adamts5, Itgb1, Vtn) and collagen deposition (e.g., Col5a1, Col5a3). We also examined chromatin architecture through Hi-C experiments and discovered that restoration of CTCF levels remodels the global architecture of chromatin to closely resemble healthy myocyte chromatin. This finding supports the hypothesis that rescue of CTCF levels ameliorates cardiac disease by reverting the observed pathological gene expression profile through genomic reorganization. In summary, our project highlights the crucial role of CTCF in heart failure and demonstrates that structural repair of chromatin organization through modulation of CTCF levels is a novel and realistic approach to treating heart failure.
Abstract P1145: All-in-one AAV CRISPR/dCas9VPR Vectors Enable Endogenous Gene Activation In Cardiomyocytes
Authors: Schoger Eric , Stengel Laura , Riedl Dominic , Lehmann Lorenz H , Hinkel Rabea , Sossalla Samuel , Zelarayan Laura C
Published: October 2023

The transcriptional landscape of cardiomyocytes harbors the cellular adaptation upon stress, but remains out of scope for therapies preventing heart failure progression. CRISPR/Cas9 mediated endogenous gene activation (CRISPRa) using enzymatically inactive Cas9 (dCas9) fused to transcription activation domains (VPR) allows for precise and fine-tuned gene activity control when targeted to promoter or enhancer regions of interest via guide RNAs (gRNAs). Transgenic mouse models and human induced pluripotent stem cell (hiPSC) lines were established for proof-of-concept (PoC) gene activation studies in cardiomyocytes. However, the size of the commonly used Streptococcus pyogenes Cas9 exceeds the packaging limits of gene therapy relevant adeno-associated virus (AAV), hampering the therapeutic use of the technology. We aimed to engineer AAV vectors based on smaller, enzymatically inactive Cas9 variants fused to a minimalized VPR-transactivation domain co-expressing a single gRNA for all-in-one gene activation strategies. We rendered mini-Cas9s enzymatically inactive by mutating the evolutionary conserved RUVC and HNH regions and C-terminally attached a minimal VPR domain as well as a HA-tag. As PoC, we expressed mini-dCas9VPR under the CMV promoter in HEK293T cells and confirmed expression of the Cas9 variants via immunoblotting. When we targeted mini-dCas9VPR to the anti-hypertrophic transcription factor KLF15 promoter region or the PoC target POU5F1 promoter, we could confirm significant gene activation compared to non-transfected and non-targeted gRNA controls (KLF15: 3-fold change, POU5F1: 4-fold change, n ≤ 4). We next exchanged the CMV promoter by the TNNT2 promoter to allow cardiomyocyte-specific expression of mini-dCas9VPR. Upon targeting the KLF15 promoter with these vectors transfected in hiPSC-cardiomyocytes, we observed significantly elevated KLF15 mRNA levels compared to controls (1.5-fold change, n ≤ 4) and robust expression of the mini-CRISPRa system, respectively. In conclusion, we present novel vectors for AAV mediated cardiomyocyte-specific endogenous gene regulation which are currently tested in hiPSC-based models, patient-derived cardiac slices and for in vivo gene activation.
Abstract P1149: Junctophilin-2 Promotes Cardiomyocyte Survival By Blocking Murf1-mediated Junctin Ubiquitination And Proteasome-dependent Degradation
Authors: Ji Xiaoyun , Huang Yifan , Ni Rui , Fan Guo-Chang , Zheng Dong , Jones Douglas L , Song Long-Sheng , Chakrabarti Subrata , Su Zhaoliang , Peng Tianqing
Published: October 2023

Background:
Junctophilin-2 is required for the development, maturation and integrity of the t-tubule system and the gating stability of RyR2 in cardiomyocytes. This study investigated whether and how junctophilin-2 maintained junctin, a scaffold protein stabilizing RyR2, to prevent cardiomyocyte death under stress.

Methods:
Cardiomyocytes were exposed to conditions of stress including palmitate, doxorubicin, or hypoxia/re-oxygenation. Adenoviral vectors were employed to manipulate expression of junctophilin-2 and junctin in cardiomyocytes. Molecular/cellular/biochemical analyses were conducted.

Results:
Different conditions of stress decreased junctophilin-2 expression through aberrant autophagy and concomitantly induced a reduction of junctin protein in cardiomyocytes. Over-expression of junctophilin-2 preserved the protein levels of junctin and attenuated cytosolic Ca
2+
and apoptosis in cardiomyocytes under stress. Knockdown of junctophilin-2 reproduced the detrimental phenotypes of stress in cardiomyocytes. Notably, over-expression of junctin prevented cardiomyocyte death under stress whereas knockdown of junctin offset the protective effects conferred by junctophilin-2 over-expression. Mechanistically, junctophilin-2 blocked MURF1-junctin interaction thereby preventing junctin ubiquitination and proteasome-dependent degradation. Mass spectrometry analysis identified multiple ubiquitination sites on the junctin protein and the non-ubiquitinated junctin mutant (K8R/K102R/K107R/K140R) was resistant to degradation.

Conclusions:
This study uncovers an unrecognized role of junctophilin-2 in preventing junctin ubiquitination and degradation in maintaining cytosolic Ca
2+
homeostasis. Junctophilin-2 and junctin represent two new survival factors of cardiomyocytes and thus, may be new therapeutic targets for cardiac protection.
Abstract P1155: P62 Overexpression Rescues Defective Autophagy Flux In Cardiomyocytes Deficient In P21 Activated Kinase 1
Authors: Geffken Shawn , Wun Tint T , Guha Anoushka , Jaiswal Pooja , Ahmed Kinza , Sooknanan Caleb , Kobayashi Tamayo , Kobayashi Satoru , Liang Qiangrong
Published: October 2023

P21-Activated Kinase 1 (PAK1) is a serine-threonine kinase that plays a critical role in cardiomyocyte survival under numerous stressful conditions. However, the underlying mechanisms for PAK1’s cardioprotective effects remain unclear. Autophagy is a lysosome-dependent degradation pathway for eliminating and recycling long-lived proteins and unwanted cellular components, which is essential for cellular homeostasis. In this study, we investigated whether and how PAK1 regulates autophagy in cardiomyocytes. PAK1 was either silenced by siRNA knockdown or overexpressed by adenovirus-mediated gene transfer in H9c2 cardiac myoblast cells. Autophagy flux was determined by measuring LC3-II protein levels or by a fluorescent autophagy reporter with and without the lysosomal protease inhibitors pepstatin A (pepA) and Aloxistatin (E64d). PAK1 downregulation reduced LC3-II levels in the total cell lysates, which were not affected by pepA and E64d, suggesting that knockdown of PAK1 inhibited autophagy flux. Inversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 was sufficient to accelerate autophagy flux. Interestingly, the expression of p62, a ubiquitin-binding autophagic adaptor, was concurrently upregulated by PAK1 overexpression or downregulated by PAK1 knockdown, suggesting that p62 may mediates PAK1-dependent autophagy. Indeed, overexpression of p62 restored autophagy flux in the absence of PAK1 (Figure 1). Together, these results suggest that PAK1 is both sufficient and necessary for maintaining normal autophagy function in cardiomyocytes, which is likely mediated by p62.
Abstract P1171: Golgi Localized DHHC3 And DHHC7 Contribute To The Initiation Of The Cardiac Hypertrophy Response In Vivo
Authors: Baldwin Tanya A , Brody Matthew J , Kuwabara Yasuhide , Zhang Weiqi , Molkentin Jeffery D
Published: October 2023

Lipid modifications provide a layer of control over cardiac signaling molecules, including small GTPases and heterotrimeric G-proteins. Lipidation of signaling factors often permits their preferential localization to intracellular membranes and organelles where they then activate downstream effectors. S-palmitoylation is a reversible lipid modification catalyzed by a family of 23 palmitoyl acyltransferases, which are characterized by a conserved zinc finger-like aspartate-histidine-histidine-cystine (zDHHC) domain. Although the importance of GTPase palmitoylation is appreciated, our understanding of enzyme-substrate specificity remains uncertain and how these enzymes regulate cardiac stress signaling and hypertrophy is unknown. To examine the roles of the cardiac-expressed zDHHCs, we overexpressed zDHHC3, 5, 6, 7 and 13 using adeno-associated viruses. Overexpression of membrane-localized zDHHC5, ER localized zDHHC6 or Golgi-localized zDHHC13 had no effect on cardiac function, while overexpression of zDHHC3 or zDHHC7 (both Golgi-localized) caused dilated hypertrophic cardiomyopathy. zDHHC3 and zDHHC7 expression were also upregulated in the heart in response to transverse aortic constriction. To gain a better understanding of the physiological role of zDHHC3 and zDHHC7 in the heart we utilized gene-deleted mice subjected to models of pathological hypertrophy. At baseline, neither single (zDHHC3 and zDHHC7) or double (zDHHC3/7) knockout mice exhibit alterations in cardiac function. Total levels of GTPase signaling proteins are unchanged, although both single and double knockout mice have reduced palmitoylation of Gαs and Rac1. In response to pressure overload, knockout of both zDHHC3/7 delayed hypertrophic progression at an early timepoint (1 week), although the response is unchanged at a late timepoint (8 weeks). We conclude that both zDHHC3 and 7 contribute to the initiation of cardiac hypertrophy but are not key regulators of hypertrophic signaling.
Abstract P1183: Phosphorylation Signatures Of Human Heart Failure: Characterizing Dilated Cardiomyopathy-associated Signaling Pathways At The Cardiomyocyte Intercalated Disc
Authors: Reitz Cristine J , Tavassoli Marjan , Kim Da Hye , Shah Saumya , Lakin Robert , Teng Allen , Zhou Yu-Qing , Li Wenping , Hadipour-Lakmehsari Sina , Backx Peter , Emili Andrew , Oudit Gavin Y , Kuzmanov Uros , Gramolini Anthony
Published: October 2023

Heart failure remains a highly prevalent condition with diverse etiology, yet the underlying signaling mechanisms are not fully understood. Despite the profound effects of post-translational protein modifications on downstream signaling, limited studies have investigated the cardiac phosphoproteome in human heart failure. We hypothesized that a combined proteomic and phosphoproteomic analysis of human dilated (DCM) and ischemic (ICM) cardiomyopathy would reveal novel etiology-associated disease pathways. Integrative analyses of left ventricular explants from DCM patients (
n
=4) vs. non-failing controls (
n
=4), and left ventricular infarct vs. non-infarct, and peri-infarct vs. non-infarct regions of ICM patients (
n
=4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. Each pair-wise comparison revealed shared and etiology-specific signatures, with a unique DCM-associated enrichment of cell-cell adhesion pathways. We focused our attention on αT-catenin (CTNNA3) as a cardiomyocyte intercalated disc candidate phosphoprotein with a unique cluster of 4 hyperphosphorylated sites in DCM hearts (
P
<0.0001). Overexpression of non-phosphorylatable hCTNNA3 in
ex vivo
isolated adult mouse cardiomyocytes showed internalized protein expression and weaker cell-cell adhesion vs. wildtype (WT) and phospho-mimetic forms. We established an
in vivo
mouse model using recombinant adeno-associated virus 9 (rAAV9) harboring hCTNNA3-WT, hCTNNA3-phospho-null, or empty rAAV9 control. Phospho-null CTNNA3 mice developed left ventricular dilation and contractile dysfunction (% EF; 51.25±1.17 phospho-null vs. 62.07±1.20 WT vs. 66.76±1.42 empty;
n
≥10) with impaired left ventricular conduction velocity (cm/s; 36.83±1.10 phospho-null vs. 47.74±2.04 WT;
n
=6), by echocardiography and
ex vivo
optical mapping. Loss of CTNNA3 phosphorylation led to intercalated disc remodeling with internalization and dissociation of CTNNA3, connexin 43, N-cadherin, β-catenin, and plakophilin 2 from the adherens junction, using high-resolution confocal imaging. Together these findings reveal a compensatory role for αT-catenin phosphorylation in maintaining cardiomyocyte intercalated disc organization in human DCM.
Abstract P2002: Variability In The Relationship Between Heart Rate And Automatic Breathing Rate Throughout The Mouse Lifespan
Authors: Gime Suri , Ahmet Ismayil , Morrell Christopher , Lakatta Edward G
Published: October 2023

Background:
Autonomic neural surveillance maintains a symphonic relationship between heart and breathing rhythms. We have established how heart beat interval variability changes across mouse lifespan (6 months - 30 months) in the presence and absence of double autonomic blockade (Baltimore Longitudinal Study on Aging in Mice, BLSAM; Geroscience 2022, PMID: 35759167).

Objective:
To determine how the automatic breathing rate (BR) changes with age in the same BLSAM cohort, and whether the BR and Heart Rate (HR) track each other in the presence and absence of double autonomic blockade across the lifespan of individual mice.

Methods:
The BLSAM cohort consists of 58 mice in which HR and BR were assessed from the EKG (under light anesthesia) at 3-month intervals, beginning at 6 months of age until the end of life (30 months of age). EKG was recorded in the presence and absence double autonomic blockade (i.p injection of atropine, 0.5mg/kg, and propranolol, 1mg/kg). Data were analyzed via a mixed ANOVA.

Results:
The BR remained stable until approximately 21 months of age, and then became sharply reduced between 21 and 30 months of age (Fig. A). The pattern of HR change over the adult life span was similar to that of BR (Fig. B). Both HR and BR were significantly (p<0.05) reduced in the presence of double autonomic blockade (arrows in figs). The relative magnitude of BR reduction beyond 21 months of age was substantially greater than the relative reduction in HR over this age range, resulting in a marked increase in HR/BR Ratio (Fig. C), which was also significantly impacted by autonomic function.

Conclusion:
Thus, beginning at approximately the 70% of maximum mice lifespan, the harmony between heart rhythm and breathing rhythm gradually deteriorates.
Abstract P2003: Cardiomyocyte Polyploidy: Friend Or Foe?
Authors: Chakraborty Archan , Wolf Matthew , Fox Donald
Published: October 2023

Developmental polyploidization (whole genome duplication) of cardiomyocytes is well conserved across mammals, birds and
Drosophila
. However, it is unclear whether precise control of cardiomyocyte polyploidy is essential. We found that there is a chamber-specific asymmetry to cardiomyocyte polyploidy in the
Drosophila
cardiac organ which is regulated by differential sensitivity of Insulin signaling. Higher ploidy cardiomyocytes correspond to the heart chamber, while lower ploidy cardiomyocytes correspond to the aorta chamber. Using immunofluorescence staining (IF), optical coherence tomography (OCT) and live imaging, we found that reducing chamber-specific ploidy affects cardiac function and reduces the velocity of circulating hemocytes, which indicates systemic heart failure. Intriguingly using human donor heart samples, we found that chamber specific asymmetry in cardiomyocyte ploidy and insulin sensitivity is similar to
Drosophila
. Our findings suggest that precise control of ploidy levels for cardiac organ sculpting is conserved across species. Polyploidization may have negative effects on cardiomyocyte regeneration, but our results suggest that region-specific control of ploidy levels is important for organ development and functionally.
Abstract P2004: T Lymphocytes Promote Diastolic Dysfunction And Fibrosis In A Non-Hypertensive Angiotensin II Mouse Model Of Heart Failure
Authors: Kim Michelle M , Mezzaroma Eleonora , Martinez Naya Nadia , Mauro Adolfo G , Alcaide Pilar , Abbate Antonio , Toldo Stefano A
Published: October 2023

Background:
Angiotensin (Ang) II signaling and diastolic dysfunction are hallmarks of heart failure (HF). Ang II promotes hypertension (HTN)-mediated cardiac fibrosis and remodeling as well as T lymphocyte activation during the pathogenesis of HF. We hypothesized that AngII induces HF, independent of HTN, through T cell activation in a mouse model.

Methods:
Using a non-HTN AngII model, we administered AngII (0.2 μg/kg/day) or H
2
O vehicle through subcutaneous pumps in wild type (WT) or T cell receptor α knockout (
Tcrα
-/-
; bereft of T cells) mice (15 wks old, N=5-7/group). We used flow cytometry to detect T cells (CD45+CD3+) in LV single-cell suspensions. LV dimensions, ejection fraction (EF), and diastolic function (isovolumetric relaxation time - IRT) were measured after 4 wks with Doppler echocardiography. LV end-diastolic pressure (EDP) was measured with a Millar catheter. Heart slides were stained with picrosirius red to measure interstitial fibrosis.

Results:
T cells were significantly increased in the LV by AngII vs H
2
O (23.0±2.1% vs 9.4±1.7%, p<0.001). All groups had similar LV dimensions and EF (not shown). AngII increased IRT (15.1±1.0 vs 8.6±0.6 ms, p<0.0001) and LVEDP (9.0±1.3 vs 3.2±0.5 mmHg, p<0.001) in WT mice vs H
2
O, but did not increase IRT and LVEDP in Tcrα-/- mice (11.8±0.8 vs 9.5±0.8 ms and 3.2±0.8 vs 2.9±0.5 mmHg; all p=ns), which were significantly lower than that of WT mice (all p<0.05). AngII increased interstitial fibrosis in WT mice (2.2±0.2 vs 0.7±0.1%, p<0.0001) while Tcrα-/- hearts had significantly less fibrosis (1.0±0.1%, p<0.001).

Conclusions:
T cells are increased in the LV and promote diastolic dysfunction and fibrosis in a non-HTN AngII HF mouse model.
Abstract P2005: Mechanosensitive Piezo-1 Channel Aggravates Ischemia Induced Adverse Cardiac Remodeling And Dysfunction
Authors: Umbarkar Prachi , Ruiz Ramirez Sulivette , Ejantkar Suma , Tousif Sultan , Toro Cora Angelica , Galindo Cristi L , Zhang Qinkun , Lal Hind
Published: October 2023

Background:
Mechanotransduction is a physiological process through which mechanical forces are sensed and converted to biological responses. It is fundamental for morphogenesis during early development and organ functions during adult life. An injured heart undergoes dynamic phases of structural and functional remodeling, exposing cardiac cells to the fluctuating mechanical environment. Piezo-1 (PZO1) is a recently discovered mechanosensitive ion channel with widespread physiological importance. However, its role in the pathophysiology of heart disease is not fully understood.

Methods and Results:
The qPCR analysis confirmed that PZO-1 expression increases in cardiomyocytes (CM) post-MI. To evaluate the role of CM-PZO1 in MI-induced adverse cardiac remodeling, conditional CM-specific PZO1 KO mice were generated using tamoxifen-inducible α-MHC promoter driven MerCreMer transgene. At 12 weeks of age, mice were subjected to a well-established tamoxifen diet protocol followed by MI surgery. Cardiac function was monitored by serial echocardiography. In the control group, EF and FS declined from 2 weeks post-MI, indicating systolic dysfunction. These changes were associated with the development of structural remodeling as reflected by a significant increase in LVIDs, HW/TL ratio, CM CSA, and hypertrophy markers (ANP, BNP). Interestingly, all these parameters were essentially normalized in CM-PZO1 KO mice, confirming that CM-specific PZO1 deletion prevents the progression of pathological hypertrophy and systolic dysfunction in MI heart. Moreover, CM-PZO1 KO displayed significant alterations in gene expression of ECM proteins such as connective tissue growth factor (CTGF), latent TGF-β binding protein 2 (LTBP2), cartilage oligomeric matrix protein (COMP), cartilage intermediate layer protein (CILP) while collagen and fibronectin levels were comparable between control and KO groups. These observations indicate a potential role of CM-PZO1 in modulating cardiac ECM and scar properties under pathological conditions.

Conclusion:
Our findings suggest that PZO1 serves as a key cardiac mechanotransducer under pathological condition and represent a novel therapeutic target for treating heart diseases.
Abstract P2008: Contractile Adaptation Of The Right Ventricular Myocardium In Pulmonary Hypertension
Authors: Mehdi Rana Raza R , Neelakantan Sunder , Wang Eric , Zhang Peng , Choudhary Gaurav , Avazmohammadi Reza
Published: October 2023

Introduction:
Pulmonary hypertension (PH) is characterized by increased right ventricular (RV) afterload inducing notable biomechanical remodeling in the RV free wall (RVFW) myocardium. While changes in the passive behavior of the RVFW have been studied, its contractile adaptations in PH remain to be investigated.

Methods:
Two sugen-hypoxia models were developed by injecting SU5416 into Sprague Dawley (SD) and Fischer (CDF) rats (n=4 each) and placing them in a hypoxia chamber for 3 weeks, followed by 1 week of normoxia. Wild-type rats (n=4), injected with vehicle and kept in normoxia, were used as controls. Right heart catheterization was perfomed at week 4. RVFW samples were skinned in 10% Triton. Samples were activated at pCa 4.5. Mechanical tests were performed isometrically at 20% stretch. Circumferential and longitudinal stresses were summed to report ensemble stress.

Results:
RV systolic pressure and stroke volume showed significant increase and decrease in both PH groups, respectively, compared to controls (Figs. 1A, B). While increases in initial stress, representing the passive behavior, did not reach significance in either PH group (Fig. 1C), total stress, representing the combined passive and active behavior, increased significantly only in CDF-PH group (Fig. 1D), known to exhibit a more maladaptive RV remodeling compared to SD-PH. Select stresses are shown in Figs. 1E, F.

Conclusions:
While elevated passive stresses in the RVFW are commonly reported in PH, the measurement of contractile stresses is essential to properly assess the adaptation of the RV. Further studies are needed to determine the role of passive vs. active stresses in the outcome of RV remodeling.
Abstract P2016: Extracellular Stiffness Induces Glucose Dysregulation Via Microtubule-dependent Mechanism
Authors: Vite Alexia , Eaton Deborah , Bouhrira Nesrine , Bedi Kenneth C , Margulies Kenneth B
Published: October 2023

Previous studies have reported profound changes in metabolic fuel utilization pathways during the development of heart failure, including changes in glucose uptake, glucose transporter mislocalization and/or Insulin-resistance. insulin stimulates glucose transport in muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Multiple reports have shown that the microtubules in adipocytes and muscle cells play an important role in the insulin-stimulated glucose transport process. We recently demonstrated, using a tunable stiffness substrate mimicking disease myocardium, that we could induces early, cell-autonomous remodeling of adult cardiomyocytes that is dependent on detyrosination of α-tubulin. We therefore employed this culture substrate to define how adult isolated rat cardiomyocytes glucose uptake response is to Insulin when cultured on stiff vs. soft. At the same time, we studied the localization and expression of Glut4 transporter. We found that 48h on stiff substrate was sufficient to induce a loss of insulin sensitivity in our isolated cardiomyocytes. Immunofluorescence reveals a decrease of Glut4 at the Intercalated disc (ID) but show increase of aggregates of the Glut4 transporter at the cell surface. Western Blot analyses shows no change in the total expression of the transporter. Finally, knowing the importance of microtubules network in the trafficking of Glut4, we overexpressed of tubulin tyrosine ligase (TTL) to prevent the detyrosination of microtubules. Surprisingly, TTL not only restored Glut4 at the ID and decreased the number of aggregates, but it also significantly increased glucose uptake as well as the total amount of Glut4 by more than 2-fold compared to soft control. In conclusion, we demonstrated the importance of microtubule dynamics in cardiomyocytes for the Insulin-dependent trafficking of Glut4 to the membrane.
Abstract P2020: Binding Of Filamin C With Actin Is Essential For Cardiac Development And Function
Authors: Zhou Xiaohai , Fang Xi , Ithychanda Sujay S , Wu Tongbin , Gu Yusu , Wang Li , Bogomolovas Julius , Qin Jun , Chen Ju
Published: October 2023

Filamin C (FLNC), a filamin family member predominantly expressed in striated muscles, is crucial for bridging the extracellular matrix and the cytoskeleton in cardiomyocytes (CMs), and the maintenance of heart integrity and function. FLNC consists of an N-terminal actin-binding domain (ABD) and 24 immunoglobulin (Ig)-like repeat domains at C-terminus. Many pathogenic mutations on N-terminal ABD have been found in patients with cardiomyopathies. However, little is known about how the actin-binding activity intrinsically contributes to the function of FLNC in mammalian hearts. Using structure-based design, we mutated two key actin binding residues F93 and L98 into Ala and Glu respectively in FLNC ABD (F93A/L98E) and show that the mutations completely disrupted the FLNC-actin interaction. We then generated a knock-in mouse model, demonstrating that loss of FLNC-actin interaction in embryonic CMs led to embryonic lethality and cardiac developmental defects displayed by ventricular rupture and reduced CM proliferation. Moreover, disruption of FLNC-actin interaction in adult CMs resulted in severe DCM phenotypes and reduced survival rate, accompanied with aberrant levels of key cytoskeleton components. These phenotypes resemble those of FLNC knockout mouse models, demonstrating a vital role of the FLNC-actin binding in the function of CMs. Our results also provide a foundation for understanding the mutation-induced cardiomyopathies associated with dysregulation of FLNC ABD-actin interaction.
Abstract P2025: Aminoacyl-trna Synthetase Complex-interacting Multifunctional Protein 3 (aimp3) Is A Key Regulator Of Cardiac Homeostasis
Authors: Das Anindhya S , Rabolli Charles P , Martens Colton , Accornero Federica
Published: October 2023

In eukaryotes, a group of aminoacyl-tRNA synthetases evolved to form a complex thought to facilitate protein synthesis by efficiently providing charged tRNAs to the translating ribosome. The human muti-synthetase complex consists of 8 aminoacyl-tRNA synthetases and 3 non-enzymatic proteins known as aminoacyl-tRNA synthetase complex-interacting multifunctional proteins (AIMP1, 2, and 3). Aside from their role in translation, these multifunctional proteins are involved in other biological processes such as inflammation, apoptosis, and DNA damage response. However, their functions in specialized cells such as cardiomyocytes have not been studied. Here, we report that cardiomyocyte-specific conditional knockout (cKO) of AIMP3 in mice leads to lethal cardiomyopathy, which presents with severe systolic dysfunction, fibrosis, and inflammation. Cardiomyocytes from AIMP3 cKO mice show no change in global protein synthesis, suggesting that AIMP3 plays a critical translation-independent role. Proteomics and oxygen consumption analyses reveal a defect in mitochondrial activity and energy metabolism with ablation of AIMP3. Altogether, we highlight the importance of AIMP3 for the preservation of heart function.
Abstract P2028: Uncoupling Endothelial Trpv4 Mechanotransduction Attenuates Pressure Overload-induced Cardiac Hypertrophy Via Rho/yap/vegfr2 Mediated Coronary Cngiogenesis
Authors: Thodeti Charles K , Adapala Ravi K , Katari Venkatesh , Ohanyan Vahagn A , Paruchuri Sailaja M
Published: October 2023

Left ventricular hypertrophy (LVH) is a bipolar response, starting as an adaptive response to the hemodynamic challenge, but over time progressing towards a maladaptive pathology with a loss of ventricular function. Despite the profound influence on cardiac function, the mechanotransduction mechanisms that regulate myocardial angiogenesis, leading to heart failure, are not well known. Here, we demonstrate that endothelial-specific deletion of mechanosensitive ion channel TRPV4 preserved cardiac function and structure and reduced fibrosis via increased capillary density compared to WT mice, 28 days post-transverse aortic constriction (TAC). Interestingly, comprehensive RNA sequencing analysis revealed an upregulation of pro-angiogenic factors (FGF2, NOS3, and VEGFα) in TRPV4
ECKO
hearts after TAC compared to TRPV4
lox/lox
. Further, an increased expression of VEGFR2 and activation of the YAP pathway was observed in TRPV4
ECKO
hearts. Mechanistically, we found that downregulation of TRPV4 in human ECs induced matrix stiffness-dependent activation of YAP and VEGFR2 via the Rho/Rho kinase/LATS pathway. Our results thus suggest that endothelial TRPV4 acts as a mechanical break for coronary angiogenesis, and uncoupling TRPV4 mechanotransduction attenuates pathological cardiac hypertrophy by enhancing coronary angiogenesis.
Abstract P2039: Alteration In Intercalated Disc Composition Impacts Cardiac Disease Progression
Authors: Raabe Janice , Stathopoulou Konstantina , Wittig Ilka , Fielitz Jens , Cuello Friederike
Published: October 2023

Intercalated discs (IDs) connect cardiomyocytes electro-mechanically and ID dysfunction has been associated with the onset of cardiomyopathies and arrhythmias. The ID- and myofibril-associated protein nebulin-related anchoring protein (NRAP) was identified as a putative novel interaction partner of protein kinase D1 (PKD). PKD1 phosphorylates class IIa histone deacetylases (HDAC), leading to their cytosolic export. This causes a de-repression of HDAC-mediated inhibition of myocyte enhancer factor 2 (MEF2)-dependent fetal and pro-hypertrophic gene expression. We investigated the regulation of NRAP by PKD1 in the developing and diseased heart. PKD activity was modulated by using the pharmacological PKD inhibitor BPKDi and/or the pro-hypertrophic stimulus phenylephrine (PE) in neonatal rat ventricular myocytes (NRVMs) and -derived engineered heart tissues (EHTs). NRVMs were analyzed by immunofluorescence, western blotting and stable isotope labelling with amino acids in cell culture/mass spectrometry. The outcome was studied by exposure to an autophagy (bafilomycin A1) or a proteasomal (MG132) inhibitor or performing proteome analysis from hearts of MuRF-1/3 double-knock-out (DKO) mice. Exposure of NRVMs to PE enhanced myofibril maturation and NRAP translocation towards IDs while BPKDi led to perinuclear accumulation of NRAP and improved force in EHTs. Combined BPKDi and PE disturbed sarcomeric structure and prevented NRAP localization at the ID. Proteomic analysis revealed that BPKDi reduced NRAP abundance and enhanced the abundance of proteins involved in proteasomal, lysosomal and spliceosomal pathways. Inhibition of proteasomal protein degradation by MG132 increased NRAP levels, whereas NRAP was 42-fold more abundant in hearts of MuRF-1/3-DKO mice that show a protein surplus cardiomyopathy. In conclusion, PKD1 impacts NRAP expression and translocation, and negatively affects EHT force development while proteasomal inhibition increased NRAP abundance suggesting a regulation via the PKD1-HDAC-MEF2 axis. Further investigations aim to study pathways involving PKD1 to regulate myofibril assembly and maintenance in the development and progression of cardiac disease.
Abstract P2042: Linking Extracellular Matrix Stiffness With Cardiomyocyte Structure And Function
Authors: Bowers Stephanie L , Baldwin Tanya , Grimes Kelly , York Allen , Sargent Michelle , Kuwabara Yasuhide , Meng Qinghang , Johansen Anne Katrine K , Molkentin Jeff D
Published: October 2023

The composition and organization of the extracellular matrix (ECM) provides cues to myocardial cells that direct normal function and responses to stress. However, little is known about how exactly a change in environmental tension leads to alterations in cardiomyocyte cell size and contractile force. Collagen I comprises the majority of cardiac ECM where it is crosslinked and matured by the lysyl oxidase (LOX) family of enzymes that includes LOX and LOX-like (LOXL) proteins. LOX and LOXLs are frequently upregulated in the heart after injury, and their inhibition has been shown to alleviate increases in tissue stiffness. Cardiac injury models result in activation and recruitment of many cells that secrete growth factors and modify the ECM, which makes it difficult to address specifically how cardiomyocytes are responding to changes in matrix tension alone. Therefore, we utilized LOX or LOXL overexpression as a tool to stiffen the ECM in the absence of pathophysiologic injury and paracrine factors. Transgenic overexpression of LOX increases myocardial stiffness and results in mild cardiac dysfunction by 9 months of age. While younger adult mice do not display a phenotype at baseline, they have an exaggerated response to pressure overload hypertrophy. In a different set of experiments, adeno-associated viruses (AAV9) for LOXL1, -2, or -3 were administered to mouse pups via cardiac injection at perinatal day 7. By 12 weeks of age, LOXL1 overexpression leads to cardiac hypertrophy with preserved ejection fraction, while increased LOXL2 or LOXL3 results in decreased cardiac function (each with differential patterns of myocardial fibrosis). Ongoing studies are assessing how these functional changes are associated with alterations in tissue stiffness and cardiomyocyte behavior. Determining how cardiomyocytes respond to alterations in the physical properties of the ECM will improve our understanding of the etiology of progressive cardiomyopathies and fibrosis.
Abstract P2043: Loss Of Full-length Mylk3 Causes Dilated Cardiomyopathy Via A Myl2-independent Mechanism
Authors: Williams Jack L , Smith Christopher J , Mastroianni Giulia , Stewart Michelle , Cutillas Pedro , Tinker Andrew , Metherell Lou
Published: October 2023

Introduction:
We recently discovered C57BL/6N male mice develop dilated cardiomyopathy (DCM) at 12 months, while the closely related C57BL/6J do not. We identified a variant (Chr8:85365179A>T, c.-5T>A) in myosin light chain kinase 3 (
Mylk3
) in the B/6N substrain that abolishes translation of the predominant transcript (ENSMUST00000034133). MYLK3 phosphorylates many sarcomere proteins, in particular myosin light chain 2 (MYL2), vital for cardiac development and function. We therefore hypothesise this variant drives the DCM in the B/6N mice.

Results:
To determine whether the
Mylk3
variant alone can cause DCM in mice, we generated two novel isogenic mouse lines. The first carries the
Mylk3
c.-5T>A variant on the C57BL/6J background. The second carries the ‘wildtype’
Mylk3
variant on the C57BL/6N background (c.-5A>T, C57BL/6N
Mylk3.6J
). While wildtype male C57BL/6J mice developed normally, male littermates expressing the mutant
Mylk3
allele (C57BL/6J
Mylk3.6N
) show signs of DCM at just 4 weeks old, with increased left ventricular diameter and volume, thinning of the posterior wall and a reduced ejection fraction. In contrast, the C57BL/6N mice develop DCM symptoms, but the C57BL/6N
Mylk3.6J
mice have smaller left ventricles, thicker left ventricular posterior wall and elevated ejection fraction. We assessed MYL2 phosphorylation by western blot, and contradictory to previous reports, found no difference between any of our mice lines. This may be due to the persistence of a shorter
Mylk3
isoform that is unaffected by the variant due to a distinct N-terminus (ENSMUST00000122452; D3Z630). There was no difference between the isoforms in their ability to phosphorylate MYL2 suggesting the shorter isoform could compensate. Phosphoproteomics identified over 500 differentially phosphorylated peptides in 3-day old C57BL/6J and C57BL/6J
Mylk3.6N
mice. These include many key cardiac transcription factors, such as NFATC2 and FOXK1.

Conclusion:
We hypothesise that loss of MYLK3 may cause dilated cardiomyopathy in our mice by a MYL2-independent mechanism, possibly by altering vital cardiac transcription causing altered heart development.