bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract GS.07: Cardiac Bridging Integrator 1 Gene Therapy Rescues Heart Failure In Swine.
Authors: Li Jing , Balmaceda Pia , Ha Thuy , Kwan Eugene , Hitzeman Tara , Palatinus Joseph , Hoareau Guillaume , Ranjan Ravi , Shaw Robin M , Hong Tingting
Published: October 2023

Heart failure (HF) is a major cause of morbidity and mortality worldwide, yet with limited therapeutic options. Recent evidence indicates that the cardiac bridging integrator 1 (cBIN1)-organized transverse tubule microdomain is key to calcium handling and cardiac function. In rodent models, adeno-associated virus 9 (AAV9)-cBIN1 effectively reconstitutes microdomains to rescue HF. Here we explored the therapeutic efficacy of AAV9-cBIN1 in a minipig model of non-ischemic HF. 6-7 months old Yucatan minipigs were subjected to continuous right ventricular rapid pacing (RVP) at 170 bpm. After 8-11 weeks of RVP, minipigs developed dilated cardiomyopathy with increased left ventricular (LV) end diastolic volume (EDV, 69.0 ± 5.3 vs. baseline 50.0 ± 3.8 mL) and reduced ejection fraction (EF, 36.3 ± 2.9 vs. baseline 68.7 ± 0.8 %). Once EF ≤ 40% over a week, pigs were randomized to receive AAV9-cBIN1 (6 x 10
11
vg/kg, intravenous, n = 3) or control (AAV9-GFP or PBS, n = 3). Weekly echocardiography, bloodwork, clinical assessment, and pacing interrogation were performed. In control animals, LV further dilated with EF declining and all three animals died of severe HF symptoms by 7 weeks post injection (terminal EF 18.3 ± 1.7, EDV 107.4 ± 25.0). Despite of continuous RVP, AAV9-cBIN1 limited LV dilation and rescued EF (58.0 ± 4.2, 59.0 ± 4.4 % at 4, 13 weeks post injection). Two cBIN1-treated animals survived to the end of protocol at 6 months post treatment (EF, 57.5 ± 15.5 %), with the third pig currently surviving at 13 weeks post treatment with a 60% EF. No significant toxicity was observed. In conclusion, low dose intravenous cBIN1 gene therapy rescues non-ischemic HF and improves mortality in pigs.
Abstract GS.08: Cardiolipin Is A Novel Target Of Therapy To Mitigate Myocardial Injury Due To Veno-arterial Extracorporeal Membrane Oxygenation (va-ecmo)
Authors: Kapur Navin K , Swain Lija , Bhave Shreyas , Qiao Xiaoying , Reyelt Lara A , Mahmoudi Elena , Everett Kay , Warner Mary , Awata Junya , Chin Michael T
Published: October 2023

Cardiolipin (CL) is a mitochondrial specific phospholipid that maintains integrity of the electron transport chain (ETC) and plays a central role in myocardial ischemia-reperfusion injury. VA-ECMO use to provide hemodynamic support for acute myocardial infarction (AMI) has grown exponentially; is associated with poor outcomes; and is the subject of active clinical trials, yet the mechanistic impact of VA-ECMO on myocardial damage in AMI remains poorly understood. We hypothesized that VA-ECMO acutely depletes myocardial CL and promotes myocardial damage in AMI.

We first report that levels of CL and tafazzin, a key enzyme required for CL synthesis, are significantly reduced in the left ventricles (LV) of human subjects requiring VA-ECMO compared to subjects without VA-ECMO prior to heart transplantation. Six hours of exposure to VA-ECMO also decreased LV levels of CL and tafazzin in healthy swine compared to sham controls. To explore whether CL depletion by VA-ECMO increases infarct size, we performed left anterior descending artery (LAD) occlusion for a total of 120 minutes followed by 180 minutes of reperfusion in adult swine in the presence and absence of MTP-131, an amphipathic molecule that interacts with CL to stabilize the inner mitochondrial membrane. Compared to reperfusion alone, VA-ECMO activation beginning after 90 minutes of LAD occlusion increased infarct size (33±11% vs 48±7%, p<0.001). VA-ECMO also decreased CL and tafazzin levels, disrupted mitochondrial structural integrity, reduced ETC function and promoted oxidative stress. Compared to reperfusion alone or VA-ECMO before reperfusion, intracoronary delivery of MTP-131 five minutes before VA-ECMO activation reduced infarct size (22±8%, p=0.03 vs IRI and p<0.001 vs VA-ECMO). Treatment with MTP-131 also restored CL and tafazzin levels, stabilized mitochondrial function, and reduced oxidative stress in the LV.

Our results identify a novel mechanism by which VA-ECMO promotes myocardial dysfunction and further identify cardiolipin as an important target of therapy to reduce infarct size and preserve mitochondrial function for patients requiring VA-ECMO for acute myocardial infarction. Future clinical studies are required to test the translational utility of this approach.
Abstract GS.101: Cardiomyocyte DCHS2 Inhibition Protects Against Heart Failure
Authors: Li Haobo , Kang Jiayi , XIAO CHUNYANG , GUERRA JUSTIN , Freeman Rebecca , Rhee James , Rosenzweig Anthony
Published: October 2023

Loss of cardiomyocytes (CMs) and impaired mitochondrial function are major causes of heart failure (HF). We recently found that cardiac Dachsous Cadherin-Related 2 (DCHS2) is induced in pathological hypertrophy and HF but inhibited by exercise. CM DCHS2 expression in vivo causes pathological hypertrophy while its deletion induces physiological hypertrophy and increases markers of cardiomyogenesis. Whether and how DCHS2 inhibition protects against pathological hypertrophy or HF are unknown. CAS9 knock-in mice were injected with AAV9 carrying scrambled (control) or guide RNA1 (gRNA1) targeting DCHS2 exon 2 together with troponin T-driven Cre and subjected to transverse aortic construction (TAC). 10 weeks after TAC, DCHS2 knockdown (KD) in CMs reduced heart (HW) and lung weight / tibial length (TL) ratios (HW/TL: 11.14±0.76 vs 13.72±0.72 mg/mm, p=0.04, n=6/group), increased fractional shortening (40.67±3.65% vs 28.80±3.51%, p=0.04, n=6/group) and decreased CM size. DCHS2 KD in CMs increased markers of CM proliferation (Ki67 and pHH3, PCM1 double positive cells, all p<0.05) and expression of mitochondrial respiratory chain complexes I-V protein and PGC1α mRNA (all p<0.05) while attenuating TAC-induced disruption of mitochondrial structure and reactive oxygen species production measured by DHE staining. In primary CMs, DCHS2 gene KD by siRNA increased CM proliferation (13.93±1.09% vs 10.11±0.14%, p=0.03, n=6) which was prevented by Yap1 KD (13.93±1.09% in DCHS2 KD vs 7.45±0.89 in DCHS2/Yap1 double KD, p<0.01, n=6). DCHS2 KD also increased mitochondrial oxygen consumption (354.8±25.86 vs 248.2±10.42 pmol/min, p<0.01, n=6) and enhanced expression of mitochondrial biogenesis-related genes as well as mtDNA copy number (all p<0.05). These DCHS2 KD-induced changes were not affected by Yap1 KD but were canceled by KD of either PGC1α or Acadvl, which is essential for very long chain fatty acids oxidation (all p<0.05). DCHS2 pulldown demonstrated that Acadvl directly bound DHCS2 in CMs. In conclusion, DCHS2 inhibition protects against HF through Yap1-dependent cardiomyogenesis and Acadvl-dependent mitochondrial effects.
Abstract GS.12: A New Mouse Model To Investigate The Early Steps Of Arrhythmogenic Cardiomyopathy
Authors: Schinner Camilla , Xu Lifen , Castellanos-Martínez Ramón , Zimmermann Aude , Zimmermann Aude , Hanns Pauline , Kuster Gabriela M , Spindler Volker
Published: October 2023

Arrhythmogenic Cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrosis, systolic dysfunction and life-threatening arrhythmias. Mutations in genes of the desmosomal adhesion complex such as desmoglein-2 (Dsg2) are the major cause, but the underlying mechanisms leading to the disease are not well understood. Accordingly, only symptomatic treatment is available. Here, we establish an inducible Dsg2-W2A knock-in mouse model with temporal control of disease onset as new approach to analyse the early processes underlying the ACM phenotype. We have previously characterized a constitutive Dsg2-W2A knock-in model which develops a cardiac phenotype bearing all major criteria of ACM directly after birth. To control the onset of disease, we now generated a mouse line bearing the Dsg2-W2A mutation and a
loxP
-flanked Dsg2 on separate alleles, leading to functional hemizygosity of the Dsg2-W2A mutation upon tamoxifen-induced Cre expression. Detailed characterization of this iDsg2-W2A model revealed early repolarization changes in ECG recordings starting directly after induction and progression to extensive impairment of electrical conduction over 10 weeks. These changes were paralleled with the appearance of ventricular and supraventricular arrhythmias. Starting two weeks after induction, reduced cardiac output function of the right and later of the left ventricle was detectable via echocardiography. Finally, these mice developed structural changes with loss of cardiomyocytes and ventricular fibrosis similar to ACM patients. This study gives detailed insight on the early steps of ACM showing a sequence from changes in conduction, occurrence of arrhythmia to functional impairment, starting in the right and progressing to the left ventricle, together with structural changes of the heart. We expect this model to be valuable to discern patho-mechanisms in detail and to identify and test novel treatment options for ACM.
Abstract GS.13: Dworf Overexpression In The Heart Results In Enhanced Mitochondrial Function
Authors: Brito-Estrada Omar , Makarewich Cat A , Gibson Aaron
Published: October 2023

Objective:
Previously, we identified dwarf open reading frame (DWORF) as a microprotein that regulates SERCA2a in the sarcoplasmic reticulum (SR). New data from our lab indicates that a fraction of DWORF localizes to the mitochondria. The goal of these studies was to analyze the role of DWORF in the mitochondria.

Methods & Results:
To identify novel DWORF interacting proteins, we performed a BioID assay in neonatal rat ventricular myocytes (NRVMs) expressing a mTurbo-DWORF fusion protein. In addition to identifying SR protein SERCA2a, we unexpectedly identified several mitochondrial proteins. Percoll gradient fractionation of mouse heart lysates showed DWORF is distributed across SR, mitochondria associated membranes (MAMs), and purified mitochondrial fractions. Seahorse mito stress test performed in isolated mitochondria from wildtype (WT), DWORF transgenic (Tg), DWORF knockout (KO), and PLN KO mouse hearts showed that mitochondria from DWORF Tg mice have increased maximal O
2
consumption rates (OCR) while mitochondria from DWORF KO hearts have reduced OCR compared to WT. To measure mitochondrial Ca
2+
dynamics, we performed mitochondrial Ca
2+
uptake assays and found that DWORF Tg hearts have increased Ca
2+
uptake rates while DWORF KO mice have reduced Ca
2+
uptake rates compared to WT. Western blot (WB) analysis of mito extracts from WT and DWORF Tg hearts indicated elevated levels of the mitochondrial Ca
2+
uniporter (MCU), while no observable changes were detected in electron transport chain (ETC) subunit expression. WB analysis also revealed a reduction in phosphorylated pyruvate dehydrogenase (PDH) in DWORF Tg mice, indicating that the enhanced mitochondrial activity observed in DWORF Tg mice may be due to a more active TCA cycle. Additionally, metabolomics analysis shows that DWORF Tg mice have lower levels of fatty acids and carnitines than WT.

Conclusions:
DWORF overexpression leads to increased mitochondrial respiration, enhanced mitochondrial Ca
2+
uptake, and reduced PDH phosphorylation (increased PDH activity), while DWORF loss-of-function results in the opposite phenotype. Collectively, these data indicate that DWORF plays a previously unrecognized role in the mitochondria and contributes to mito Ca
2+
regulation and energetics.
Abstract P1004: Cellular Heterogeneity In The
Nkx2.5
+ Anterior Lateral Plate Mesoderm Underlies Lineage Diversity Of The Cardiovascular System
Authors: Coskun Hakan , Gao Xinlei , Chen Kaifu , Burns C. Geoffrey , Burns Caroline
Published: October 2023

The cardiovascular system comprises diverse cellular lineages that emerge from progenitor cells in the anterior lateral plate mesoderm (ALPM). In zebrafish,
nkx2.5
expression initiates in the bilateral field of ALPM progenitors during early somitogenesis before their spatial segregation into two main subpopulations. The cardiac fraction, including first (FHF) and second heart field (SHF) progenitors, migrates to the midline and gives rise to ventricular myocardium. The pharyngeal fraction remains lateral and sequesters in the cores of the pharyngeal arches (PAs). Progenitors in PA2 give rise to craniofacial muscles and three lineages in the outflow tract, while those in PA3-6 differentiate into pharyngeal arch artery (PAA) endothelium. The mechanisms underlying
nkx2.5
+ progenitor cell segregation into the various downstream fates remain unclear. Here, we demonstrate that
nkx2.5
+ progenitors in the ALPM are heterogenous with predetermined lineage outcomes. Specifically, single-cell RNA sequencing of
nkx2.5:ZsYellow
+ progenitors revealed six transcriptionally unique populations, including a cardiomyocyte cluster (FHF-derived) and a large lateral plate mesoderm (LPM) population that further subclustered into three distinct groups. LPM-1 differentially expressed
aplnra
,
hey1
, and the retinoic acid (RA)-eliminating enzyme
cyp26c1
, LPM-2 expressed
bmp4, bmp6
, and
cdh11
, and LPM-3 expressed the transcriptional repressor
prdm1a
and the RA-producing enzymes
rhd10a
and
aldh1a2
.
In situ
hybridization uncovered regionalization in the
nkx2.5
+ ALPM with LPM-1 cells occupying the anterior-medial and central region of the field and LPM-3 the posterior domain. Targeted photoconversion of
nkx2.5:Kaede
revealed that LPM-1 are PA2 progenitors while LPM-3 are progenitors for PA3-6. We are currently performing spatial transcriptomics to generate a comprehensive expression map of the ALPM, localizing and tracing LPM-2 progenitors, and performing functional studies that suggest a
tbx1
-RA-prdm1a signaling pathway is required for LPM-3 specification. In conclusion, our data highlight progenitor heterogeneity in the ALPM as a source of the lineage diversification required to build the cardiovascular system.
Abstract P1005: Parallel Identification Of Gene-Environment Interactions In Cardiac Development Using Human Induced Pluripotent Stem Cells
Authors: Nishiga Masataka , Liu Chun , Liu Lichao , Qi Lei Stanley , Wu Joseph C
Published: October 2023

Most human diseases are influenced by both genetic factors and environmental factors. However, it is not easy to identify causative combinations of genes and environmental factors that interplay in disease development. Congenital heart defects (CHDs), which is the most common birth defect occurring 1% of newborns in the US, are primarily caused by genetic mutations. On the other hand, phenotypes of CHDs are typically inconsistent among individuals with the same variants and the penetrance is often incomplete, which suggests the important role of environmental factors. Environmental factors known to be involved in the development of CHDs include maternal smoking, diabetes, inflammation, and use of certain medications. Here, we investigated gene-environmental interactions in cardiac development. First, we developed a CRISPR-screen system that screens essential genes in cardiac differentiation from human induced pluripotent stem cells (iPSCs). Our custom CRISPRi library targets
~
700 genes, which are enriched in human adult or fetal hearts. Our screen system successfully identified positive control genes such as TNNT2, TNNC1, and TBX20 as well as genes that have not been intensively investigated in human cardiac development such as BAMBI and SMAD6. Second, to identify gene-environment interactions, we used the system under the exposure to various environmental stresses such as nicotine, ethanol, inflammatory cytokines, and multiple teratogenic drugs and identified stress-specific gene-environment interactions. This approach is applicable to any other screens of gene-environment interactions.
Abstract P1012: Wnt Signaling Is Required For Myxomatous Valve Disease In A Mouse Model Of Marfan Syndrome
Authors: Xu Na , Alfieri Christina , Yutzey Katherine
Published: October 2023

Introduction:
Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome (MFS) includes valve leaflet thickening and increased immune cell infiltration. However, the underlying mechanisms by which immune cells are recruited in MFS valves remain undefined.

Hypothesis:
Inhibition of Wnt signaling prevents immune cell recruitment and MVD progression in MFS valves.

Methods:
Mice with the mutation of Fibrillin 1 (

Fbn1
C1039G/+

) recapitulate histopathological features of MFS and Wnt signaling activity was detected in TCF/Lef-lacZ reporter mice. The expression level of C-C motif chemokine ligand 2 (CCL2) was detected in

Fbn1
C1039G/+

mice by qPCR and RNAscope. Inhibition of Wnt signaling was achieved by conditional induction of the soluble Wnt inhibitor Dkk1 in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ;

Fbn1
C1039G/+

mice. Dietary doxycycline was administered for one month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for immune cells were performed.

Results:
Wnt signaling as detected by TCF/Lef-lacZ reporters and CCL2 expression were upregulated early in mitral valve disease progression, prior to immune cell infiltration in

Fbn1
C1039G/+

mice. Inhibition of Wnt signaling by Dkk1 induction at 1-month of age prevented the induction of myxomatous mitral valve disease, indicated by reduced valve leaflet thickness with decreased CD45+ leukocytes and CCR2+ immune cells in

Fbn1
C1039G/+

mice. However, later Wnt inhibition starting at 2 months when inflammation and myxomatous changes are occurring did not prevent the progression of myxomatous mitral valve disease.

Conclusions:
Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation, but is not responsible for valve disease progression once it has been initiated.
Abstract P1019: High-throughput Generation And Functional Characterization Of Myosin Heavy Chain 7 Variants In Genome-edited Cardiomyocytes
Authors: Yang Kai-Chun , Friedman Clayton , Fayer Shawn , Pendyala Sriram , Chien Wei-Ming , Loiben Alexander , Chao Leslie , Ahmed Dania , Karbassi Elaheh , Murry Charles , Starita Lea , Fowler Douglas
Published: October 2023

Hypertrophic cardiomyopathy (HCM), an inheritable cardiac condition characterized by idiopathic left ventricular hypertrophy, can cause sudden cardiac death or heart failure. Autosomal-dominant missense mutations in
MYH7
(encoding the sarcomeric βMHC protein) account for 33% of genotype-positive HCM cases. While pathogenic
MYH7
variants are clinically-actionable, 75% of
MYH7
missense variants in ClinVar are variants of unknown significance (VUS). Gene-editing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can help determine the function of
MYH7
VUS, however, the discovery of new variants by genetic testing outpaces the ability to gene-edit hiPSCs. Thus, we developed a novel method called
CR
ISPR
a
On-
T
arget
E
diting
R
etrieval (CRaTER) which increases the success of gene-editing by 25-fold compared to current methods. We leveraged CRaTER to enrich for hiPSCs edited with a library of
MYH7
missense variants and recovered 113 different heterozygous variants. As deleterious mutations can reduce protein stability, we flow sorted
MYH7
variant hiPSC-CMs into bins based on differing βMHC abundance followed by next generation sequencing of each bin to calculate variant abundance scores. This assay identified 31
MYH7
missense variants as functionally abnormal (lower abundance scores than synonymous controls), including all known pathogenic variants in the library. This βMHC depletion phenotype was validated in clonal hiPSC-CMs and supported by western blotting of myocardium from a patient with a pathogenic
MYH7
missense mutation. Furthermore, we rescued βMHC protein levels by genetically ablating the gene encoding for Muscle RING-finger protein-1, an E3 protein ligase that targets βMHC for degradation, and found that the cardiomyocyte hypertrophy induced by the pathogenic
MYH7
E848G HCM variant was significantly blunted. In summary, CRaTER enables the generation of gene-edited hiPSC lines at unprecedented scale. Functional analysis of
MYH7
variant hiPSC-CMs reveals a novel correlation between loss of βMHC protein and clinical pathogenicity that can be leveraged for clinical variant interpretation. Interventions that prevent the loss of βMHC may potentially be therapeutic for
MYH7
-associated HCM.
Abstract P1022: Synthetic Copolymer-Based Membrane Stabilizers Confer Protection To Dystrophin-Deficient Striated Muscle
In Vitro
And
In Vivo
Authors: Quick Joseph D , Hahn Dongwoo , Bauer John , Hassler Joseph , Thompson Brian , Hackel Benjamin , Lodge Tim , Bates Frank , Metzger Joseph M
Published: October 2023

Duchenne muscular dystrophy (DMD) is a chronic and progressive muscle wasting disease resulting from the inability to produce the cytoskeletal protein dystrophin. The absence of dystrophin increases the susceptibility of the sarcolemma to mechanical damage and ultimately contributes to muscle degeneration and fibrosis. A growing body of evidence suggests that synthetic membrane stabilizing copolymers can be an effective strategy to limit contraction-induced sarcolemma damage. First-in-class copolymer-based membrane stabilizer Poloxamer 188 (P188) is a linear, amphiphilic triblock copolymer demonstrated to protect dystrophic cardiac and skeletal muscle in a wide variety of
in vitro
and
in vivo
models. However, the architectural features of P188 responsible for its membrane protective effects remain to be fully elucidated. Additionally, various other membrane-stabilizing copolymers differing in their size, architecture, and composition have been useful in structure-function studies. Here we sought to elucidate the properties of synthetic copolymers that confer stabilization to the dystrophin-deficient membrane. Using newly developed techniques, we designed and investigated an innovative class of branched “bottlebrush” copolymers at the single skeletal muscle fiber and organismal levels. Our results identify an intriguing, novel bottlebrush copolymer effective at previously unreported nanomolar concentrations
in vitro
. We also show in preliminary studies that bottlebrush copolymers may further confer protection during isoproterenol-induced cardiac stress testing in dystrophin deficient mice
in vivo
. These findings will be discussed in terms of copolymer-based membrane-interfacing molecules in the context of stabilizing dystrophin-deficient striated muscle.
Abstract P1023: Nonsense Mutations In Atrial Myofilament Regulatory Protein MyBP-HL Are Subject To Proteolytic Degradation And Loss Of Functional Protein
Authors: Alvarez-Arce Alejandro A , Wittenkeller Lucas , Burnham Hope , Barefield David Y
Published: October 2023

Background:
Myosin binding protein-H like (MyBP-HL) is a recently identified atrial myofilament protein encoded by the gene MYBPHL. The Arg255Stop mutant has been linked to atrial enlargement, atrial fibrillation, and dilated cardiomyopathy. Deletion of MYBPHL in mice recapitulates this human phenotype. MyBP-HL is composed of two Immunoglobulin domains (Ig) and one Fibronectin domain (FnIII), like the structure and sequence of the last three C-terminal domains (C8-C9-C10) of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations fail in myofilament incorporation and result in a rapid degradation of the truncated protein and are a major cause of hypertrophic cardiomyopathy. There are 9 premature stop mutations in MYBPHL identified in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307).

Hypothesis:
We hypothesize that MYBPHL nonsense mutations will share a common mechanism that leads to MyBP-HL failing to integrate into the sarcomere, resulting in truncated MyBP-HL degradation and loss myofilament incorporation.

Results:
We demonstrate heterologous expression of MyBP-HL in neonatal rat cardiomyocytes can incorporate in a similar sarcomere pattern as cMyBP-C. Expression of the nine nonsense mutations do not localize into the sarcomere. Neonatal rat cardiomyocytes transfected with MyBP-HL constructs were treated with proteasome and/or calpain inhibitors. We observed that wild-type MyBP-HL and MyBP-HL truncating mutations are degraded mainly by the proteasome, although we also identified a significant calpain involvement in MyBP-HL degradation. Shorter MyBP-HL truncated peptides are degraded as a poisoning peptides, but longer peptides are not degraded.

Conclusion:
Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament as are subject to degradation. The differential expression of short vs. long truncating MyBP-HL mutants suggest a differential pathogenic mechanism for subsets of these mutations.
Abstract P1029: Abnormal Progenitor Cell Differentiation And Cardiomyocyte Proliferation In Hypoplastic Right Heart Syndrome
Authors: Zhao Mingtao , Yu Yang , Ye Shiqiao , Garg Vidu , xu zhaohui
Published: October 2023

Pulmonary atresia with intact ventricular septum (PA-IVS) is a rare (4-8 per 100,000 live births) type of hypoplastic right heart syndrome (HRHS) where the right-sided structures in the heart are malformed. In PA-IVS, the pulmonary valve that acts to regulate the unidirectional flow of blood from the right ventricle (RV) to the lungs does not open, resulting in no connection between the RV and the pulmonary arteries. After surgical or catheter-based intervention, PA-IVS patients have clinical outcomes that range from single-ventricle palliation to a biventricular repair. Mechanisms underlying the spectrum of RV hypoplasia in PA-IVS are difficult to fully ascribe to an atretic pulmonary valve. There are no reliable animal models available for studying disease mechanisms of PA-IVS. In this study, we leverage patient-specific induced pluripotent stem cells (iPSCs) and single-cell RNA sequencing to elucidate cellular etiologies of ventricular hypoplasia in PA-IVS. PA-IVS iPSC-derived cardiomyocytes are less proliferative compared to controls under both static and cyclic stretch, suggesting that genetic factors may contribute to a spectrum of ventricular hypoplasia in PA-IVS. Single-cell transcriptomic analysis reveals that cell lineage determination towards second heart progenitors is suppressed, with enhanced differentiation into first heart field and epicardial lineages during cardiac differentiation. Additionally, biological pathways associated with cell proliferation and cell cycle progression are downregulated whereas mitochondrial activities are elevated in early cardiomyocytes originated from PA-IVS patients. In conclusion, abnormal cell lineage differentiation and cardiomyocyte proliferation may underlie the cellular and developmental etiologies of ventricular hypoplasia in HRHS.
Abstract P1042: Stem Cell Models Reveal Sarcomere Inhibition As A Potential Treatment For Pediatric Restrictive Cardiomyopathy
Authors: Staudt David , Serrano Ricardo , Mercola Mark
Published: October 2023

Introduction:
Pediatric Restrictive Cardiomyopathy (RCM) is a genetic cardiomyopathy resulting from profoundly increased stiffness of the wall of the cardiac ventricle with normal or low normal contractility. Treatment options are limited, with most patients necessitating heart transplant. Therapies that can improve diastolic function are needed for this severe disease. Recently, direct inhibition of the sarcomere with mavacamten, a cardiac myosin antagonist, has been shown to be effective in treating a related disease, Hypertrophic Cardiomyopathy (HCM), in adults. However, whether it would be useful for the diastolic dysfunction seen in RCM is unknown.

Hypothesis:
Treatment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from RCM patients with mavacamten will rescue diastolic deficits.

Methods:
Cardiomyocytes were differentiated from induced pluripotent stem cells (iPSCs) originally derived from a pediatric patient with RCM, as well as a paired isogenic CRISPR-corrected line. Cells were treated with increasing doses of mavacamten and contractility and resting tension were measured using a novel traction force microscopy platform.

Results:
At baseline, RCM cells showed elevated contractility, resting tension, and calcium sensitivity compared to their isogenic control. Mavacamten treatment decreased both contractility and resting tension of RCM cardiomyocytes.

Conclusions:
Mavacamten improves both systolic and diastolic derangements in an hiPSC model of RCM.
Abstract P1061: Salivary Extracellular Vesicle-associated LncRNAs As Novel Non-invasive Biomarkers For Acute Decompensated Heart Failure: The Pioneering SEAL-HF Study
Authors: Spanos Michail , Gokulnath Priyanka , Sheng Quanhu , Chatterjee Emeli , Li Guoping , Luong Kathy , Lehmann Helge Immo , Wang Ashley Mingyi , Nwokemodo-Ihejirika Chinemerem , Limpitikul Worawan , Varrias Dimitrios , Wong David T , Das Saumya
Published: October 2023

Acute Decompensated Heart Failure (ADHF) is a major global health concern, and early diagnosis is critical for effective management. Non-invasive biomarkers have the potential to improve patient outcomes substantially. In this study, we investigated the presence of extracellular vesicle (EV)-associated long non-coding RNAs (lncRNAs) in plasma of ADHF patients and explored their non-invasive detection in saliva as the foundation of the SEAL-HF study. We performed RNA-sequencing on plasma-derived EVs from ADHF patients at admission and discharge and healthy controls to identify differentially expressed lncRNAs and mRNAs, using a fold change cutoff of ±1.5 and an adjusted p-value of <0.05. Subsequent validation was carried out via qRT-PCR on plasma EV RNA samples from 182 patients. Salivary EVs were isolated and enriched, and their EV RNA content was profiled. Transcriptomic analysis unveiled several dysregulated EV-derived mRNAs and lncRNAs in ADHF patients compared to controls. Importantly, four lncRNAs (LINC00989, lnc-CALML5, AC092656.1, and RMRP) exhibited significant changes between the acute congestive phase and decongestion after diuresis and were found to circulate mainly within the EV compartment. Salivary EVs contained EV protein markers ALIX, CD81, and heart-derived protein marker Troponin T. qRT-PCR assay demonstrated the presence of LINC00989, lnc-CALML5, AC092656.1, and RMRP in salivary EVs, as well as cardiac-specific genes such as PLN. Our results highlight the potential of salivary EV-associated lncRNAs as non-invasive biomarkers for ADHF diagnosis, laying the groundwork for the SEAL-HF study and future clinical applications.
Abstract P1064: Gene Editing Technologies To Understand Knockout And Over Expression Of Circadian Rhythm Regulatory Genes In Human Derived Spheroid Model.
Authors: Mattapally Saidulu , Labarge Wesley C , Walcott Gregory , Mattapally Chandrakala , Zhang Jianyi J
Published: October 2023

Introduction:
Human induced pluripotent stem cells (hiPSCs) have the ability to produce a huge variety of terminally differentiated cell types that can be used for tissue engineering, drug research, and cell therapy. Using CRISPR/Cas9 gene-editing technologies, hiPSCs with knockout mutations for Per1, Per2, CIITA, B2M, N-Cadherin, Connexin43 and over expression of Connexin43 were created. Cardiomyocytes (CM) were created from hiPSCs, and spheroid culture was carried out as previously described. The molecular basis of circadian regulation of human induced pluripotent stem cells (hiPSCs) is still under investigation.

Methods:
From hiPSC CMs, we created and described a 3D spheroid culture. We assessed the effectiveness of spheroid transplantation for cardiac healing in immunecompromised mice and pigs. We investigated border zone macrophage markers, border zone vascular density, and cell proliferation in myocardial infarction after spheroid transplantation. We used pedigree analysis and 20 years of family health diagnostics data to analyze the clinical consequences of type 2 diabetes medications among metformin combo patients in big extended family (N=30)..

Results:
TNF-Alpha, IL1-beta, NOS2, and IL-6 relative expression were shown to be lower in the KO group than in the WT group. Significant engraftment of hiPSC-derived cardiac spheroids was seen up to 4 weeks after transplantation, according to histological examination. Arrhythmogenicity is significantly altered by the spheroid therapy. Ventricular tachycardia may be related to macrophage immunological regulation. To further understand, we looked at the effects of a medication combination of dapagliflozin and metformin in a large extended family, where 5 out of 30 members had type 2 diabetes. One patient experienced mildly tachycardic heart issues over the course of a 20-year medication treatment.

Conclusion:
Our study established the promising therapeutic potential of spheroids model from hiPSC differentiated CMs to understand Circadian rhythm gene regulation.
Abstract P1065: Primitive Macrophages Enhance The Function Of Bioengineered Human Cardiac Microtissues
Authors: Hamidzada Homaira , Pascual-Gil Simon , Laflamme Michael A , Keller Gordon , Radisic Milica , Epelman Slava
Published: October 2023

Human cardiac bioengineering has traditionally focused on cardiomyocyte differentiation and maturation. Unlike normal cardiac growth in the native human heart, human pluripotent stem cell (hPSC)- derived cardiomyocytes are typically grown in the absence of key supportive populations, which may negatively impact function due to a failure to recreate complex multicellular environments.
In vivo
murine studies have demonstrated that embryonic-derived resident cardiac macrophages promote neonatal cardiac regeneration, yet macrophages have not been incorporated in cardiac bioengineering approaches. We hypothesized that inclusion of human embryonic stem cell derived primitive macrophages (hESC-macrophages) into bioengineered human cardiac microtissues (“Biowires”) would improve cardiomyocyte functional properties. We found that addition of hESC-macrophages into Biowires led to enhanced contractile force, improved relaxation, reduced excitation threshold, increased maximum capture rate, and increased conduction velocity that mechanistically was correlated with improved calcium cycling, indicating hESC-macrophages improve a broad range of electromechanical properties. Moreover, hESC-macrophages enhanced microtissue function across cardiomyocyte cell sources and bioengineering platforms (Experimental N=6 independent cellular differentiations across 2 different platforms). These changes occurred in the absence of significant transcriptional shifts in cardiomyocytes. Rather, Biowires with hESC-macrophages had reduced mitochondrial DNA release, oxidative stress and cytotoxicity in the very early phases of tissue formation, thereby promoting a favorable environment for cardiac microtissue development. In conclusion, these data reveal a previously unappreciated, yet major beneficial role for macrophages specifically in human cardiac tissue engineering. In general, bioengineering human tissue continues to be a major research focus, and our data argue that inclusion of yolk sac-derived macrophages may improve early microtissue formation and modeling
in vitro
, and could enhance cell delivery approaches
in vivo
.
Abstract P1069: Yap Mediates Cardiac Proliferation In Tead-independent Mechanisms
Authors: Xie Bing , Steimle Jeff , Deshmukh Vaibhav , Morikawa Yuka , Meng Fansen , Martin James F
Published: October 2023

Myocardial infarction (MI) is a leading cause of death worldwide. During an MI, the contractile myocardium experiences reduced blood flow causing expansive cell death. Since adult mammalian hearts lack regenerative capacity, MI damage is permanent followed by heart failure. However, recent studies have demonstrated that the neonatal mouse heart exhibits transient regenerative potential within the first 7 days, suggesting a regulatory mechanism that prevents the adult from initiating a regenerative response to cardiac injury. The Hippo signaling pathway negatively regulates mammalian cardiac regeneration by inhibiting the activity of downstream effector YAP. However, YAP5SA, which completely bypasses Hippo pathway regulation, results in YAP hyper-transcriptional activity and heart overgrowth. In this work, we aim to investigate the TEAD-independent mechanisms of YAP in promoting cardiomyocyte (CM) division and renewal. We generated another YAP protein, which can not only skip Hippo pathway inhibition, but also lose the interaction with TEADs. We found that this modified YAP promoted CM proliferation and improved cardiac repair post MI injury. Our work will uncover novel functions of YAP and improve YAP therapeutics in myocardial infarction, finally benefiting human heart health.
Abstract P1070: In Vivo Autotransplantation Of Spleen-derived Endothelial Progenitor Cells To Promote Angiogenesis In Ischemic Sites
Authors: Chung Seyong
Published: October 2023

Since mononuclear cells were first illuminated as potential endothelial progenitor cells (EPCs), bone marrow (BM) was extracted and processed as BM-derived EPCs for autotransplantation to the ischemic sites in previous studies. Still, numerous clinical trials based on this strategy showed only marginal effects, and the small number of BM-derived EPCs was suggested as a possible limitation. Meanwhile, spleen contains abundant mononuclear cells which can inherently target the ischemic sites, and these cells can be primed by hypoxia. If only they can be primed in vivo, spleen-derived EPCs can be inherently autotransplanted to ischemic sites with various advantages such as plentiful cell number, non-invasive extraction and delivery of these cells. In this study, splenic mononuclear cells were utilized to promote angiogenesis in ischemic sites. In vivo priming of splenic mononuclear cells, their inherent targeting and therapeutic effect on ischemic sites were sequentially explored towards the 'in vivo autotransplantation'. For hypoxic priming, the effect of hypoxia and hypoxic-mimetic agent (cobalt chloride) on isolated splenic mononuclear cells were comparable in both priming methods, regarding the morphology changes and the increase in expression of EPC markers. In align with these in vitro results, both hypoxic-mimetic agent-loaded nanoparticle delivery to spleen and splenic artery ligation resulted in increased expression of EPC markers in mice spleen. Moreover, the proportion and actual numbers of the cells presenting EPC marker were significantly higher in primed splenic cells than in BM cells. After the priming, these primed cells not only maintained the inherent targeting ability but also gained the ability to promote angiogenesis. Consequently, these primed cells presented therapeutic effects as evidenced by higher expression of angiogenic markers with enhanced blood flow recovery in mouse hindlimb ischemia model, compared with untreated or BM-derived EPCs' autotransplantation for conventional strategy. For EPC-based treatment of ischemia, 'In vivo autotransplantation' is simple and non-invasive strategy which can overcome the previous limitations of insufficient cell numbers.
Abstract P1088: Biophysical Crowding Of
In Vitro
Culture Media Alters The Growth Rate And Collagen Production Of Cells Used For Seeding Tissue-Engineered Heart Valves
Authors: Henshaw Kathryn G , Joyce Angelina J , Freitas Renata C , Mayer John E
Published: October 2023

Introduction:
Ideal tissue-engineered heart valves (TEHVs) for valve replacement in congenital heart disease patients would have durability and growth potential. Endothelial colony-forming cells (ECFCs) isolated from peripheral blood are an attractive source for pre-cellularizing TEHVs with a patient’s own cells, as they are isolated non-invasively and can transform into valve endothelial and interstitial phenotypes. ECFC limitations include slow proliferation and uncertain mechanisms to control extracellular matrix (ECM) production. In other cell types, culture media “crowded” with large macromolecules limits paracrine diffusion and augments both ECM production and cell growth.

Hypothesis:
As a novel study of biophysical crowding on ECFCs, we hypothesized that media crowded with macromolecules dextran sulfate or Ficoll-400 would enhance the growth rate and collagen production of ovine ECFCs.

Methods:
ECFC lines (CD31+, α-SMA+) from n=3 animals at passages 2, 3, or 7 were cultured in media with no crowder or either Ficoll-400 or dextran sulfate at four concentrations in three separate replicates (n=10, n=120, n=384). Deposited and suspended collagen was quantified. Immunocytochemical cell scoring was quantified for vWF, VEGFR-2, CD34, vimentin, and α-SMA markers.

Results and Conclusions:
Anionic dextran sulfate (DS) caused significant dose-dependent increases in media soluble collagen, despite significantly lowering cell counts. Crowding with DS significantly increased CD34 positivity, a marker for anti-angiogenic and quiescent ECFC populations. Conversely, neutral Ficoll-400 significantly increased cell counts but did not alter media collagen or CD34 positivity. Both crowder groups had altered cellular morphology but did not cause reproducible differences in positivity for endothelial markers vWF and VEGFR-2 or interstitial markers vimentin and α-SMA. In conclusion, biophysical crowding of ECFC media alters cell count and morphology, CD34 positivity, and collagen production, which may allow for fine-tuning of ECFC properties when seeding onto TEHVs. Future studies will address the different effects of neutral and negatively charged crowders and how crowders impact cellular phenotype and growth on valvular scaffolds.
Abstract P1097: Foxk1 Is A Regulator Of Cardiomyocyte Proliferation And Heart Regeneration
Authors: Sierra-Pagan Javier , Larson Thijs , Das Satyabrata , Ma Xiao , Alexy Tamas , li qinglu , Stan Patricia , Gong Wuming , Zhang Jianyi J , Garry Mary G , Garry Daniel J
Published: October 2023

While the adult mammalian heart has limited regenerative potential, the neonatal mouse heart has a remarkable regenerative capacity. Our goal focused on defining new factors and mechanisms that could enhance repair and regeneration in the adult mouse heart. FOXK1 is a transcription factor that regulates cell cycle kinetics, myogenic stem cell proliferation and skeletal muscle regeneration. During development, its expression is restricted to progenitors, somites and the heart. However, its role in neonatal heart regeneration is unknown and warrants investigation. Here, we describe a novel role for FOXK1 as a regulator of cardiomyocyte (CM) proliferation and regeneration. Using computational analysis of single nucleus RNAseq datasets of neonatal CMs, we determined that FOXK1 was expressed in CMs. qPCR analysis of P1 hearts from control or
Foxk1
null mice demonstrated that the absence of FOXK1 led to the dysregulation of
Ccne1
and
p21
expression, suggesting that FOXK1 could play a role in cell cycle kinetics in the neonatal heart. Control or
Foxk1
null mice were pulsed with EdU which demonstrated that the absence of FOXK1 led to a decrease in CM proliferation in vivo. We then engineered a transgenic mouse model that overexpressed FOXK1 using the muscle creatine kinase (
MCK-Foxk1
) promoter and isolated and cultured control or
MCK-Foxk1
CMs (72 hrs), and determined that overexpression of FOXK1 led to a significant increase in CM proliferation. Furthermore, EdU pulsing studies in adult control or
MCK-Foxk1
mice demonstrated a significant increase in EdU incorporation in adult CMs of
MCK-Foxk1
unperturbed mice compared to control. We then performed LAD ligation surgeries in control and
MCK-Foxk1
adult mice. Echocardiography 2 months following LAD ligations showed a significant increase in both ejection fraction and fractional shortening in
MCK-Foxk1
mice compared to controls. Single nucleus RNAseq analysis of control and
MCK-Foxk1
adult hearts highlighted two distinct CM populations by UMAP analysis, with the latter demonstrating a higher expression of myoblast proliferative pathways. These results identify FOXK1 as a regulator of CM proliferation and heart regeneration by employing both knockout and transgenic mouse models.
Abstract P1103: A Combinatorial Stem Cell Therapy Using HiPSC-derived Cardiomyocytes And HiPSC-derived Mesenchymal Stem Cells For Cardiac Regeneration
Authors: Sridharan Divya , Baseeruddin Alvi Syed , Mergaye Muhamad , Forehand Abbey , Ahmed Uzair , Khan Mahmood
Published: October 2023

Introduction:
Myocardial infarction (MI) results in the death of nearly a billion cardiomyocytes in the heart. While conventional therapies alleviate the injury to some extent, they do not induce myocardial regeneration. On the other hand, transplantation of stem cells and their derived differentiated cells like hiPSC-derived cardiomyocytes (hiPSC-CMs) and hiPSC-derived mesenchymal stem cells (hiPSC-MSCs) have the potential to promote myocardial regeneration by direct (replacing dead cardiomyocytes) or indirect (paracrine) mechanisms, respectively.

Hypothesis:
We hypothesized that following co-transplantation of hiPSC-CMs and hiPSC-MSCs in ischemic hearts, hiPSC-MSCs will release paracrine factors to promote angiogenesis, reduce fibrosis, and improve cardioprotection and engraftment of transplanted hiPSC-CMs.

Results:
We cultured hiPSC-CMs and hiPSC-MSCs at different ratios, 1:1, 1:2 and 1:3, respectively. We observed improved contractility (MEA, and calcium transients) in the hiPSC-CM + hiPSC-MSC co-cultures, as compared to single cell-type cultures. Additionally, following exposure to hypoxia (1%O
2
), we observed increased survival of hiPSC-CMs when co-cultured with hiPSC-MSCs (TUNEL assay). Furthermore, intramyocardial transplantation of (a) 1.5 х 10
6
hiPSC-CMs, (b) 1.5 х 10
6
hiPSC-MSCs or (c) 1.5 х 10
6
hiPSC-CMs + hiPSC-MSCs (3:1) in a mouse MI model, resulted in an improved cardiac function, LVEF (p < 0.01) and FS (p<0.01) in all animals receiving cell transplantation as compared to MI only group. Additionally, we observed efficient engraftment (immunostaining of human-specific markers, cTnT and HNA) of transplanted hiPSC-CMs in both hiPSC-CM-only and hiPSC-CM + hiPSC-MSC groups up to 4 weeks post-MI. We also observed increased neoangiogenesis (α-SMA, vWF) near the graft in the hiPSC-MSC-only and hiPSC-CM + hiPSC-MSC groups, as compared to control and hiPSC-CM-only groups. Lastly, a significant reduction in fibrosis (Masson-trichrome staining) was seen following transplantation of hiPSC-CMs + hiPSC-MSCs, compared to the control animals.

Conclusion:
Overall, our results established that a combinatorial stem therapy using hiPSC-CMs and hiPSC-MSCs for cardiac regeneration post-MI.