bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 392: Metabolic Signaling Mechanisms Governing Heart Regenerative Capacity
Authors: Zhang Song , Dzeja Petras
Published: July 2021

Energy metabolism and metabolic signaling circuits orchestrate cell commitment to self-renewal, lineage specification, differentiation and regeneration. When energy resources are plenty, cell can grow, proliferate and regenerate, however when energy is low - augmented adenylate kinase (AK)-mediated AMP signaling turns on AMPK which silences p53/p21/cyclin cell cycle metabolic checkpoint and halts cell division. Using mouse neonatal hearts, with high and low regenerative capacity, we have determined metabolomic profiles and dynamics of phosphotransfer circuits using 18O-phosphoryl labeling mass-spectrometric and 18O-assisted 31P NMR techniques. We demonstrate that loss of heart regenerative capacity after birth is associated with marked changes in heart AK-catalyzed phosphotransfer flux and AMP signaling along with changes in expression levels of p21, cyclin A and E and thymidine kinase. It appears, that in adult heart increased expression of AK isoforms (AK1, AK2 and AK1β) and augmented high energy phosphoryl and AMP signal dynamics is misread by AMPK-sensor as "low energy" state inducing blockade of cell cycle metabolic checkpoint and cardiomyocyte proliferation and renewal. Using AK-GFP constructs and immunocytochemistry we further demonstrate the distribution of AK1, AK1β, AK2 and AMPK between cytosol and nucleus and association with mitotic spindle and cytokinetic apparatus during cell division cycle. AK1 translocation to the nucleus, however, doesn’t occur in adult cardiomyocytes deficient in cytokinesis. Protein knockdown using siRNA indicates that AK2 is critical for cardiomyocyte mitochondrial biogenesis and network formation. Furthermore, we have discovered that deficiency of the AK2 isoform, which is localized in mitochondria intermembrane-intra-cristae space, arrests developmental programming and is embryonically lethal in mice. The uncovered shift in metabolic signaling mechanisms opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.
Abstract 397: Generation of Raf1 Mutant and Crispr-cas9 Corrected Isogenic iPSC-derived Cardiomyocytes to Model Hypertrophic Cardiomyopathy in Noonan Syndrome
Authors: Jaffré Fabrice , Wang Gang , Roberts Amy , Pu William , Hahn Andreas , Kontaridis Maria
Published: July 2021

Background:
Hypertrophic cardiomyopathy (HCM) is a major cause of death in infants and children. Noonan Syndrome (NS), an autosomal dominant RASopathy disorder, is characterized by multiple defects, including short stature, facial dysmorphia, and congenital heart defects that include HCM. RASopathies are caused by germ-line mutations that affect the canonical RAS-MAPK pathway. Indeed, 95% of NS patients with a mutation in
Raf1
, a gene that plays an integral role in this signaling cascade, exhibit HCM. However, the molecular mechanisms that elicit HCM in these patients remain poorly understood.

Objective:
To generate human NS
Raf1
induced-pluripotent stem cells (iPSCs), correct the mutation by genome editing and subsequently differentiate isogenic iPSC lines into cardiomyocytes to characterize the molecular and genetic basis of HCM in NS patients.

Results:
We generated iPSCs from skin fibroblasts obtained from a NS pediatric patient with a single point mutation in the
Raf1
gene. Using electroporation of four episomal vectors containing the Yamanaka factors, we obtained several iPSC clones with normal karyotypes and strong expression of pluripotent markers (Nanog, Oct4, Lin28, Sox2) as detected by RT-qPCR and immunofluorescence. We next corrected the mutation in the NS
Raf1
iPSCs using genome editing CRISPR-Cas9 nickase technology. Correction of the
Raf1
mutation was determined at the clonal level by PCR followed by Restriction Fragment Length Polymorphism and confirmed by Sanger sequencing. In addition, by inducing a Cas9 nickase-dependent frame shift mutation we also generated an isogenic iPSC line where
Raf1
gene was knockout (KO), as demonstrated at the RNA level by RT-qPCR and at the protein level by Western Blot. We next differentiated these multiple iPSC lines (mutant, corrected and KO) into isogenic beating cardiomyocytes, with more >98% of the cells positive for specific cardiomyocyte markers (α-actinin and cardiac TroponinT).

Conclusion:
We have successfully generated human NS
Raf1
isogenic iPSC lines and corresponding cardiomyocytes. Currently, we are in progress of characterizing these cardiac cells to determine the molecular basis of NS-dependent HCM. Ultimately, our work should reveal new targets to treat HCM in NS patients.
Abstract 4: Porcine Parthenotes as a Model to Evaluate Developmental Potential of Human Inducible Pluripotent Stem Cells
Authors: Koyano-Nakagawa Naoko , Dutton James , Garry Mary G , Garry Daniel J
Published: July 2021

The use of human induced pluripotent stem cells (hiPSCs) has tremendous potential for regenerative medicine by providing an unlimited source of personalized cells. A number of protocols have been established for efficient differentiation of hiPSCs to the desired lineage in vitro, such as cardiomyocytes and blood. However, the field lacks an in vivo system to evaluate the differentiation potential and quality of hiPSCs. Developmental potential of stem cells derived from experimental animals can be readily assessed by generating blastocyst chimeras and examination of the contribution to the embryos, or by the potential of teratoma formation. However, this is not possible in the case of humans. As a potential solution for this issue, we examined whether porcine parthenotes could be used as an experimental model to test the developmental potential of the hiPSCs. Parthenotes are generated by electrical activation of the oocytes collected at the abattoir and will develop up to gestational day 53 if transferred to a pseudo-pregnant sow. The embryonic culture conditions have also been established and the zygotes can develop normally to the expanded blastocyst stage (day 7 post fertilization/activation), in vitro. We took advantage of this in vitro system and examined the ability of hiPSCs to proliferate and integrate into the parthenogenetic embryos. Parthenogenetic embryos were injected with ten undifferentiated hiPSCs at day 4 (8 cell ~ morula stage) and cultured up to 72 hours. During this period, parthenotes underwent blastocoel cavity formation and hatching. Cell tracing experiments demonstrated that hiPSCs proliferated and integrated into the parthenotes. They retained pluripotency marker expression during this period. hiPSCs and their derivatives were found both in trophoectoderm and embryo proper. We further observed that the hiPSCs underwent cellular proliferation and promoted developmental progression of the parthenote in vitro. In summary, the porcine parthenote model system is an efficient high throughput system to examine the developmental capacity of human stem cell populations.
Abstract 401: Shortening of Action Potential Duration With Increased Work in Contracting Rabbit Heart
Authors: Garrott Kara E , Wengrowski Anastasia , Zhang Hanyu , Rogers Jack , Kay Matthew
Published: July 2021

Studying heart function in contracting hearts at higher workloads provides crucial knowledge of cardiac performance in high-stress situations. Ratiometric optical mapping of fully loaded hearts is a novel method to study electrical activity while replicating in vivo energy consumption.

We predict that an imbalance between energy supply and demand during increased work and hypoxia will manifest in shortened action potential duration (APD), especially in the more physiologically-relevant bi-ventricular (BiV) working heart.

Rabbit hearts were perfused with oxygenated Krebs-Henseleit solution in unloaded Langendorff and fully-loaded BiV mode. Epicardial action potentials (APs) were measured using optical mapping of Di-4-ANEPPS. Excitation ratiometry using 450 and 505 nm illumination on alternate camera frames together with motion tracking removed motion artifact. Aortic pressure, left atrial preload, and LVDP was measured. A range of workloads were studied by pacing at 330, 220, and 170 ms cycle length (CL). Gradual hypoxia was induced by bubbling with N2 gas.

In Langendorff mode, the APD was 137.67±4.29 ms, 113.44±7.89 ms, and 106.44±0.44 ms at CL of 330ms, 220ms, and 170ms, respectively, while in BiV mode, the APD was 106.56±13.03 ms, 78.00±2.33 ms, and 69.33±0.77 ms. Aortic pressure dropped from NSR in BiV hearts by 1.26% at 330ms, 4.11% at 220ms, and 11.65% at 170ms CL.

Shortening of APD, independent of restitution, with increasing HR indicates an imbalance of energy supply and demand with greater workload. KATP channels are implicated. This novel method of optical mapping reveals important implications of electrophysiological changes during high-stress situations.
Abstract 406: Interaction of T Cells From Non-ischemic Heart Failure Patients With the Activated Vascular Endothelium is Dependent on Intercellular Adhesion Molecule-1
Authors: Nevers Tania A , Velazquez Francisco , Salvador Ane , Kapur Navin , Alcaide Pilar
Published: July 2021

Background:
Increasing evidence supports a role for inflammation in the pathogenesis of chronic heart failure (HF). Our previous studies demonstrate that T cell recruitment to the heart contributes to the progression of non-ischemic pressure overload induced HF. However, clinical data describing T cell dependent mechanisms contributing to the etiology of this disorder remains unknown. We hypothesized that non-ischemic HF activates human T cells resulting in increased adhesion to the vascular endothelium and recruitment to the heart through mechanisms involving specific endothelial adhesion molecules.

Methods and Results:
We used T cells from non-ischemic HF patients and non-HF controls as well as left ventricular (LV) tissue from end stage HF after LV assisted device (LVAD) support. Using FACS analysis we found that systemic T cells are significantly elevated in HF patients compared to controls (p<0.05), including Th1, Th17, and Treg cells. Immunohistochemistry analysis revealed that CD3+ and CD4+ T cells infiltrate the LV of HF subjects which were not observed in control. To evaluate the mechanisms through which T cells interact with the vasculature and potentially infiltrate the heart, we used in vitro real time video microscopy under shear flow conditions and found that T cells from HF patients firmly adhered to TNF[[Unsupported Character - Symbol Font ]] activated HUVECS. Further analysis indicated that HF T cells adhered to ICAM-1, but not to VCAM-1 in higher numbers than control T cells. The surface expression levels of the integrin ligands for VCAM-1 and ICAM-1 (VLA-4 and LFA-1 respectively) were similar between both groups, however, HF T cells exhibited a highly polarized phenotype on ICAM-1 (p<0.005 vs control), suggesting LFA-1 is in its high affinity conformation in HF T cells.

Conclusions:
Our findings suggest that T cells are activated in non-ischemic HF and have high affinity for the activated endothelium through mechanisms involving ICAM-1- LFA-1 adhesion. Future studies will evaluate the mechanisms regulating LFA-1 activation in HF T cells and their contribution to cardiac remodeling.
Abstract 407: Modulation of Delta-Like Ligand-4 Signaling Improves Myocardial Revascularization Following Coronary Ischemia
Authors: Kumar S R , Yi Christopher , Martens Timothy , Zagger Sydney J , Duarte Antonio , Gill Parkash S
Published: July 2021

Delta-like ligand-4 (DLL4) is an arterial-specific Notch ligand and balanced signaling by DLL4 is required for functional neovascularization. We hypothesized that modest inhibition of DLL4 signaling improves myocardial revascularization following coronary ischemia. Myocardial infarction was induced by ligation of mid-left anterior descending (LAD) artery via rapid left thoracotomy in 6-8 week-old male mice with inducible endothelial-specific knockout or overexpression of DLL4. DLL4 silencing in homozygous mice on the day of or 5 days prior to LAD ligation resulted in significant reduction in revascularization. At four weeks, explanted hearts showed 1.8-fold fewer vessels in LAD territory, 3-fold greater myocardial hypoxia, 1.7-fold larger fibrotic scar and 17% increased perfusion defect in the left ventricle (LV) by scintigraphy. CT angiography confirmed fewer collateral vessels. Echocardiography showed increased LV dilation (37% higher end-diastolic volume) and 12% greater reduction in ejection fraction (EF) compared to baseline (all p<0.001). Overexpression of DLL4 showed a similarly worse outcome. Conversely, partial DLL4 knockout in heterozygous animals resulted in improved outcomes in all parameters. For translational application, animals were systemically administered 1.5mg/kg or 3mg/kg DLL4-Fc intraperitoneally three times a week beginning the day of ligation. RT-PCR analysis of downstream molecules confirmed that systemic DLL4-Fc partially inhibits Notch signaling in endothelial cells in the ischemic LV. When LAD was ligated very proximally, DLL4-Fc improved survival. With mid-LAD ligation, DLL4-Fc induced a dose-dependent increase in number of CD 31-positive vessels and by CT angiography. There was a dose-dependent reduction in hypoxic myocardial area, scar burden, and scintigraphic perfusion defect. DLL4-Fc treated mice had lower end-diastolic LV volume and preserved or improved EF. In an ischemia-reperfusion model, DLL4-Fc increased the number of vessels in the ischemic zone. Our data suggests that balanced DLL4 signaling is crucially required for myocardial angiogenic recovery following coronary ischemia. Modulation of DLL4 signaling has translational therapeutic potential.
Abstract 418: Mdm2 E3 Ligase-mediated Ubiquitination of Histone Deacetylase 1 in Vascular Calcification
Authors: Kook Hyun , Kwon Duk-Hwa , Eom Gwang Hyeon , Shin Sera , Joung Hosouk , Choe Nakwon , Nam Yoon Seok , Kook Taewon , Kim Hyung Seok , Kim Yong Sook , Koh Jeong-Tae , Kim Nacksung , Nam Kwang Il
Published: July 2021

Vascular calcification (VC) often associates with many cardiovascular and metabolic diseases. Although VC is the cause of high morbidity and mortality, molecular mechanisms have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity enhanced VC
in vivo
and
in vitro
. HDAC1 protein was reduced in cell and animal calcification models and in human calcified coronary artery and this reduction preceded VC. Calcification stresses induced MDM2 E3 ligase, which resulted in HDAC1 K74 ubiquitination. Forced expression of MDM2 enhanced VC, whereas loss of MDM2 blunted it. A decoy peptide spanning HDAC1 K74 prevented VC. These results demonstrate a previously unknown ubiquitination pathway as well as the involvement of HDAC1 in VC. Our results suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.
Abstract 426: A Novel Method to Study Viral Cardiomyopathy
Authors: Shah Millie , Borgman Cheryl , Janes Kevin
Published: July 2021

Latent coxsackievirus B3 (CVB3) cardiac infections are implicated in the development of DCM with persistent enteroviral RNA found in up to 66% (30/45) of DCM patient biopsies who are at a 6-fold higher risk of fatality. Current treatments aim at delaying heart failure but none address the viral etiology of the disease. Viral protein presence is thought to disrupt normal cellular signaling leading to tissue dysfunction. The downstream effects of viral perturbations are complex and wide-ranging; especially in proinflammatory contexts seen clinically. Thus, comprehensively understanding the molecular mechanisms of CVB3-mediated disease is key in developing treatments for viral DCM patients. In this study we built a novel in vitro model of chronic CVB3 infection to facilitate high-throughput, systems-level studies of viral cardiomyopathy.

Current methods of studying CVB3 rely on animals or animal derived cardiac cells, making large-scale intracellular signaling studies difficult, time intensive, and expensive. To facilitate high-throughput studies we used immortalized human cardiomyocytes to engineer single-cell derived cell lines that express a maturation deficient CVB3 genome. CVB3 RNA expression was validated in each line by two independent methods: gene-specific nested qRT-PCR and single molecule RNA fluorescence in situ hydridization (smFISH). Plaque-assay verified that viral RNA expression did not result in live virus release.

Microarray analysis shows that CVB3 expressing cell lines have altered immune cytokine, extracellular matrix protein, and stress-signaling protein expression. Further, differences between CVB3 expressing lines suggest differential responses to viral RNA expression which may identify a set of beneficial adaptations. Future studies will include high-throughput signaling protein activity assays we have developed specifically for phosphatase and kinase activity quantification with the aim of linking immune cytokine signaling dysregulation to pathogenic gene expression. These studies will deepen our knowledge of CVB3-mediated DCM and identify proteins whose targeted modulation could offer new treatment strategies to patients whose current options are either palliative care or heart transplantation.
Abstract 427: Micrornas Have Differential Degree of Expression in Pathological Cardiac Hypertrophy Compared to Physiological Cardiac Hypertrophy
Authors: Martinelli Nidiane C , Cohen Carolina , Silvello Daiane , Biolo Andréia , Andrades Michael , dos Santos Kátia , Clausell Nadine , Rohde Luis E , Matte Ursula
Published: July 2021

Physiological and pathological left ventricular hypertrophies (LVH) are distinct processes that have differential pattern of gene expression. Based on initial stimuli, miRs expression levels can fluctuate and then cause a variance on their targets culminating in diverse cellular pathway activation. AIM: Here we compared miRs expression between pathological cardiac hypertrophy induced by transverse aortic constriction (TAC) and physiological cardiac hypertrophy induced by voluntary exercise in running wheels (EXE). METHODS: Adult male Balb/c mice (12-14 weeks old) mice were subjected to TAC or EXE protocol and data were evaluated at 7 (TAC-7D; EXE-7D) and 35 (TAC-35D; EXE-35D) days. Hypertrophy was measured by normalizing left ventricular weight to body weight (LVW/BW). We evaluated left ventricular expression levels of miRs: -26b, 27a, -143, -150, -195 and -499 by qRT-PCR in TAC and EXE groups. Comparisons between groups were performed by ANOVA with Bonferroni correction. Results are shown as mean±SEM. Results: Sedentary and Sham groups were similar among all variables tested. Animals subjected to TAC surgery demonstrated a greater hypertrophy than EXE animals at both time points (7D: 16% vs. 7%; 35D 26% vs 12%, p<0.05 for both). MiR-26b had increased levels in TAC group at both time points (7D: 1.14±0.1 vs 0.6±0.01; 35D: 4.8±1.4 vs 1.17±0.12; p<0.01 for both). We only detected an increase in miR-27a levels in TAC-7D compared to EXE-7D (2.7±1.0 vs 0.78±0.1, p <0.05). We identified an augmentation in miR-143 levels in TAC group at both time points (7D: 1.1±0.1 vs 0.75±0.1; 35D: 1.42±0.2 vs 0.9±0.1; p<0.05 for both). We detected an increase in miR-499 levels at both time points in TAC group (7D: 4.1±0.5 vs 0.67±0.2, p<0.001; 35D: 2.2±0.4 vs 0.9±0.2, p<0.01). We found an increase in miR-195 levels only in TAC-35D group compared to EXE-35D (2.6±0.3 vs 0.9±0.1, p<0.05). We did not notice any change in miR-150 levels neither at 7 days nor at 35 days. Conclusions: These preliminary data demonstrate a differential degree of miR expression between physiological and pathological hypertrophy. Further studies comparing physiological and pathological cardiac hypertrophy are necessary to find out the turning point that deviates heart from adaptive to maladaptive growth.
Abstract 431: ScaRNAs Regulate Cardiac Differentiation and Development by Fine Tuning the Spliceosome
Authors: Bittel Douglas C , Rongish Brenda , Kibiryeva Nataliya , Filla Michael , Marshall Jennifer , Artman Michael , O’Brien James E , Johnson Rajasingh
Published: July 2021

Alternative splicing (AS) of mRNA adds diversity to the proteome and precise regulation of AS is essential for proper development. We recently showed that multiple genes critical for heart development are irregularly spliced in the right ventricle of babies with tetralogy of Fallot (TOF). We also observed reduced expression of several noncoding small cajal body associated RNAs (scaRNAs). scaRNAs direct the biochemical modification of spliceosomal RNAs and are essential for the stability and function of the spliceosome. Our results provide compelling evidence for a direct role of scaRNAs in regulating splicing of genes that are critical for heart development. To further explore this novel paradigm of developmental regulation, we analyzed the transcriptome of stem cells as they differentiated to beating cardiomyocytes. We observed significant alternative splicing with respect to timepoint (with Bonferroni correction and fdr of 5%) in 3,165 of 20,301 (15.6%) total genes. Importantly, there were 79 alternatively spliced genes among 213 genes (37.1%) known to be critical for heart development. This is a significant enrichment in the cardiac network genes (p<0.001). Most of the alternative isoforms are known protein coding variants. In addition, we saw changes in expression of several scaRNAs. scaRNA1 is reduced in the right ventricle of children with TOF and we targeted it for knockdown in the quail embryo model system. Preliminary results revealed dramatic alterations in cardiac morphogenesis and embryonic lethality. At higher levels of the antisense oligo, cells appeared to undergo apoptosis, aggregated inappropriately and failed to gastrulate. Lower concentrations resulted in initiation of gastrulation with some cells from the cardiac lineage traversing the embryonic milieu to contribute to the heart and other organs. Interestingly, these cells appeared to lack protrusive phenotypes, and may be passively moved by neighboring groups of non-electroporated motile cells. Taken together, our results suggest scaRNAs are necessary to maintain the fidelity of the spliceosome and thus play an important role in vertebrate heart development. These observations provide additional new insights into regulatory mechanisms underlying cardiac development.
Abstract 433: Disrupting the Interaction Between CaM Kinase II and Histone Deacetylase 4 - an Epigenetic Therapy for Heart Failure?
Authors: Backs Johannes , He Tao , Lehmann Lorenz H , Schmidt Andrea , Beckendorf Jan , Lewis Joe , Askoxylakis Vasileios , Katus Hugo A
Published: July 2021

CaM Kinase II (CaMKII) critically drives adverse cardiac remodeling. During the process of remodeling, CaMKII binds and phosphorylates Histone Deacetylase 4 (HDAC4), resulting in activation of the transcription factor MEF2. However, it remained unclear whether binding between CaMKII and HDAC4 causes heart failure and whether this interaction represents a novel therapeutic target. We used mouse genetics, HDAC4-based peptides and chemical biology to address these questions. First, we generated CaMKII-resistant HDAC4 mutant mice (CrH) by replacing Arg-598 (corresponds to Arg-601 in humans) of HDAC4 with Phe, because we found Arg-598 to be essential for the CaMKII-HDAC4 interaction. CrH were protected from cardiac dysfunction, hypertrophy and fibrosis in response to both pathological pressure overload or ischemia/reperfusion injury. CrH showed reduced CaMKII binding and less MEF2 activation. These data provided a proof-of-principle that the disruption of the CaMKII-HDAC4 interaction may have therapeutic potential. Thus, in a second step we engineered an HDAC4-derived peptide with homology to the CaMKII binding domain of HDAC4. This peptide competed with HDAC4 for binding with CaMKII, resulting in decreased MEF2 activation and attenuated agonist-induced cardiomyocyte hypertrophy. These data encouraged us to carry the translational pipeline one step further and we screened for small molecules that disrupt the CaMKII-HDAC4 interaction in an in vitro ALPHAScreen Assay (medium-throughput format using 78000 compounds). After a stringent validation process, 38 compounds showed > 40% inhibition. Out of these, 13 compounds effectively inhibited MEF2 activity in a cell-based assay without obvious signs for cellular toxicity, providing now potential cell permeable drug-like candidates. Chemical optimization and in vivo validation strategies are currently ongoing. In summary, we show that the CaMKII-HDAC4 interaction contributes to the development of heart failure and we identified drug-like molecules that specifically disrupt this protein-protein interaction. These findings lay the ground for a novel epigenetic therapeutic approach to combat heart failure.
Abstract 69: Reduction of Diastolic Dysfunction by Treatment of a Combination of Agonists of Adenosine Receptor in Spontaneously Hypertensive Rats Submitted to Myocardial Infarction
Authors: Zapata-Sudo Gisele , Frazão Tais N , da Silva Jaqueline S , Barreiro Eliezer J , Fraga Carlos A , Sudo Roberto T
Published: July 2021

Introduction:
This work investigated the cardioprotective actions of the combination of a positive inotropic agent (LASSBio-294 ) and a potent vasodilator (LASSBio-897) in spontaneously hypertensive rats (SHR) submitted to myocardial infarction (MI).

Methods:
Twenty four SHR (180-200 g) were randomly divided in sham-operated (SO) and infarcted groups (MI) and each group subdivided in two: treatment with vehicle (DMSO) or with LASSBio-294 + LASSBio-897 (5mg/kg each, p.o.) during 8 weeks. After treatment period, the animals were submitted to echocardiography to determine the anterior wall thickness (AWT), ejection fraction (EF), fractional shortening (FS) and the ratio of early and late transmitral filling velocity (E/A). In addition, the following hemodynamic parameters were evaluated: mean blood pressure (MBP), left ventricular end diastolic pressure (LVEDP), left ventricular end-systolic pressure (LVESP) and LV contractility and relaxation (dp/dt
max
). Hypertrophy was measured using the relation between heart weight to body weight (HW/BW). The volume fraction of collagen (%) was determined by measuring the area of H&E stained tissue within a given field.

Results:
MI induced in SHR promoted a decrease in AWT; EF; FS and E/A from 2.0 ± 0.4 to 1.6 ± 0.9 mm; from 53.1 ± 7.5 to 25.3 ± 6.4 %; from 40.0 ± 0.9 to 25.3 ± 11.0 %; and from 1.4 ± 0.1 to 0.9 ± 0.1, respectively. Treatment with the combination of drugs, increased AWT to 2.5 ± 0.6 mm; EF to 73.2 ± 1.0 %; FS to 43.5 ± 6.6%; and E/A to 1.3 ± 0.1. Increase of LVEDP from 4.6 ± 0.3 to 30.0 ± 3.6 mmHg and duplicated oxygen consumption were observed in MI-SHR. The negative dP/dt was reduced from 6152 ± 1015 to 3957 ± 1225 mm Hg/s. After treatment, all hemodynamic parameters were restored to values similar to SO group. Mean blood pressure which was increased after MI from 168. 2 ± 18.6 to 197.7 ± 10.7 returned to 137.0 ± 19.3 mm Hg after treatment. Increased deposition of colagen from 15.1 ± 3.9 to 24.0 ± 0.9 % induced by MI was prevented with treatment with the combination of drugs (12.9 ± 3.8 %). Conclusion: Oral administration of the combination of LASSBio-294 and LASSBio-897 could be considered promising in preventing cardiac dysfunction in SHR submitted to MI.
Abstract 71: Soy Isoflavone Protects Myocardial Ischemia/reperfusion Injury Through Increasing Endothelial Nitric Oxide Synthase and Decreasing Oxidative Stress in Ovariectomized Rats
Authors: Xie Liping , Meng Guoliang , Ji Yong
Published: July 2021

Rationale:
There is a special role for estrogens in preventing and curing cardiovascular disease in women. Soy isoflavone (SI), a soy-derived phytoestrogen, is a group of biologically active plant substances with chemical structures which are similar to that of an endogenous estrogen-estradiol.

Objective:
We ought to elucidate possible mechanism of SI to improve myocardial ischemia/reperfusion (MI/R) injury in ovariectomized rats.

Methods and Results:
Female SD rats were underwent bilateral ovariectomy or sham ovariectomy. One week later, rats were randomly divided into several groups and began to feed soy-free chow: sham ovariectomy operation (control group), ovariectomy with MI/R or ovariectomy with sham MI/R. Other ovariectomy rats were given different doses of SI dissolved in 0.5% carboxymethycellulose (CMC-Na) by gavage. Additional ovariectomy rats were administrated with the same volume of CMC-Na by gavage or 50 μg/kg·d of 17β-estradiol (E
2
) by subcutaneous injection. After four-week treatment, they were exposed to 30 minutes of left coronary artery occlusion followed by 6 or 24 hours of reperfusion. SI treatment decreased body weight, increased estradiol level and uterus weight. Isoflavone administration significantly reduced myocardial infarct size, improved left ventricle function and restored endothelium-dependent relaxation function of thoracic aortas after MI/R in ovariectomized rats. SI also decreased creatine kinase and malonaldehyde in plasma and attenuated oxidative stress in the myocardium. Meanwhile, SI increased phosphatidylinositol 3 kinase (PI3K) / Akt/ endothelial nitric oxide synthase (eNOS) signal pathway.

Conclusion:
Soy isoflavone protects myocardial ischemia/reperfusion injurys in ovariectomized rats through increasing PI3K/Akt/eNOS signal pathway and decreasing oxidative stress.
Abstract 74: The Role of Fibroblast-specific Canonical Tgfβ Signaling in Cardiac Fibrosis
Authors: Khalil Hadi , Kanisicak Onur , Correll Robert N. , Sargent Michelle , Molkentin Jeffery D.
Published: July 2021

Heart failure is a progressive disease characterized by cardiomyocyte loss, interstitial fibrosis, and chamber remodeling. During physiological conditions cardiac fibroblasts contribute to the homeostatic maintenance of myocardial structure as well as the maintenance of biochemical, mechanical and electrical properties of the heart. Injury and/or cytokine stimulation activate fibroblasts which transdifferentiate into myofibroblasts. These newly formed cells secrete extracellular matrix (ECM) for wound healing and tissue remodeling through their contractile activity. Fibrosis mediated by these cells can initially be a beneficial response that acutely scarifies areas after an infarct to prevent wall rupture. However, during chronic disease states such as heart failure, persistent recruitment and activation of fibroblasts leads to excessive deposition of ECM that results in stiffening and pathological remodeling of the ventricles. During chronic heart disease, cardiomyocytes, immune cells and fibroblasts secrete the cytokine transforming growth factor-TGFβ, which activates fibroblasts and promotes their conversion to myofibroblasts. Manipulation of TGFβ by losartan, which antagonizes angiotensin II (AngII) and aspects of TGFβ signaling, has shown some anti-fibrotic effects in cardiovascular remodeling. Also deletion of Tgfbr1 (type I TGFβ receptor) in cardiomyocytes or a TGFβ blocking antibody reduced the fibrotic response after pressure overload. However heart failure was not improved because deleterious TGFβ signaling in fibroblasts persisted. We therefore utilized a novel fibroblast-specific inducible Cre-expressing mouse line (Periostin-MerCreMer) to examine the canonical (Smad2/3) TGFβ signaling within fibroblasts to determine how these cells and their activation mediate disease in heart failure. Our data indicate that fibroblast-specific deletion of Smad3 but not Smad2 was sufficient to significantly inhibit myocardial fibrosis. Smad2/3 double nulls were also generated and analyzed, as were TGFBR1 and TGFBR2 loxp targeted mice, also crossed with the Postn-MerCreMer knockin allele to achieve specificity in activated fibroblasts.
Abstract 76: Effects of Long-term Angiotensin-II Infusion on Cardiac and Renal Fibrosis are Blunted in TNFR1-deficient Mice
Authors: Mayr Magdalena , Duerrschmid Clemens , Lee Dorellyn B , Medrano Guillermo , Taffet George E , Entman Mark L , Haudek Sandra B
Published: July 2021

Background:
Brief systemic infusion of Angiotensin-II (Ang-II) to wild-type (WT) mice initiates the development of cardiac interstitial fibrosis. Genetic deletion of tumor necrosis factor receptor 1 (TNFR1) obviates this development and concurrently inhibits Ang-II-induced cardiac remodeling and dysfunction. We now investigated long-term effects of Ang-II on the heart, kidney, and cardiorenal function.

Methods:
WT and TNFR1-KO mice were infused with 1.5 ug/kg/min Ang-II for 1 and 6 weeks (no uninephrectomy or high-salt diet). Heart, kidney, and serum were isolated and evaluated by histology, cytometry, qPCR, and ELISA techniques. Cardiac function was determined by 2D-echocardiography, systolic blood pressure by tail-cuff plethysmography.

Results:
Brief infusion of Ang-II to WT mice did not evoke a fibrotic response in the kidney. However, after 6 weeks, WT kidneys developed minimal, but significant interstitial collagen deposition which was supported by upregulation of collagen-I, collagen-III, and alpha-smooth muscle actin gene activation. This fibrotic development was associated with the appearance of myeloid fibroblast precursors, pro-inflammatory M1 and pro-fibrotic M2 cells, and myofibroblasts. Transcriptional expression of pro-inflammatory and pro-fibrotic genes was also increased. These changes were not seen in Ang-II-infused TNFR1-KO kidneys. In WT hearts, despite the disappearance of myeloid cells, cardiac fibrosis persisted throughout the 6-week infusion. WT hearts developed clear evidence of accelerated cardiac hypertrophy and remodeling associated with impaired systolic function. Again, these changes were not seen in Ang-II-infused TNFR1-KO hearts. By contrast, both WT and TNFR1-KO mice responded identically with similar elevations of systolic blood pressure, and serum blood urea nitrogen and creatinine levels.

Conclusions:
Ang-II-infusion induced an immediate fibrotic response in the heart while fibrosis in the kidney developed slowly. The cardiac fibrosis was accompanied by progressive adverse remodeling and worsening of function over time. TNFR1-KO mice were protected from the Ang-II-induced cardiac and renal fibrosis, despite similar increases in blood pressure and renal dysfunction.
Abstract 80: Activation of Nrf2 by Sulforaphane via the AKT/GSK-3β/Fyn Pathway Prevents Angiotensin II-induced Cardiomyopathy
Authors: Xin Ying , Bai Yang , Jiang Xin , Zhou Shanshan , Wang Yuehui , Wintergerst Kupper A , Tan Yi , Cui Taixing , Cai Lu
Published: July 2021

Aims:
Sulforaphane (SFN) as a nuclear factor erythroid 2-related factor 2 (Nrf2) activator protects the heart from, and deletion of the Nrf2 gene exaggerates, the effects of diabetes. Angiotensin II (Ang II) plays a critical role in the development of diabetic cardiomyopathy; therefore, whether SFN prevents Ang II-induced cardiomyopathy through activation of Nrf2 was examined.

Methods and Results:
The chronic cardiac effects of Ang II with and without SFN were examined in wild-type mice, transgenic Nrf2 knockout (Nrf2-KO) mice, and mice in which cardiac tissue overexpressed Nrf2 (Nrf2-TG). The signaling pathways of SFN-mediated Nrf2 activation were examined in H9C2 cells. Administration of a subpressor dose of Ang II to WT mice induced cardiac oxidative stress, inflammation, remodeling and dysfunction, all of which could be prevented by SFN treatment, which also up-regulated Nrf2 expression and activation. Nrf2-TG mice showed resistance and Nrf2-KO mice displayed resistance to Ang II-induced cardiomyopathy. Meanwhile, the ability of SFN to protect against Ang II-induced cardiac damage was lost in Nrf2-KO mice. Up-regulation and activation of Nrf2 by SFN is accompanied by activation of AKT, inhibition of glycogen synthase kinase (GSK)-3β, and increased nuclear accumulation of Fyn. In vitro up-regulation of Nrf2 by SFN in H9C2 cells was abolished and nuclear Fyn accumulation was increased when cells were exposed to a PI3K inhibitor or GSK-3β-specific activator.

Conclusion:
Nrf2 plays a central role in the prevention of Ang II-induced pathological effects, and SFN can prevent Ang II-induced cardiomyopathy through activation of Nrf2 partially via the AKT/GSK-3β/Fyn pathway.
Abstract 91: Interdependence of Mitochondrial Fission and Mitophagy in Adult Mouse Hearts
Authors: Song Moshi , Burelle Yan , Dorn Gerald W
Published: July 2021

The role of Drp1-mediated mitochondrial fission in normal hearts is controversial. Cardiomyocyte mitochondria are hypo-dynamic, but mitochondrial dynamism factors are abundantly expressed. Cardiac-specific Drp1 gene deletion (KO) provokes mitochondrial enlargement, MPTP-dependent cardiomyocyte loss, and dilated cardiomyopathy (Song et al Cell Metab, 2015). We postulated that Drp1-dependent mitochondrial fission is essential for triage and elimination of damaged cardiomyocyte mitochondria by Parkin-mediated mitophagy. Others described no benefit of Parkin KO in mice with perinatal cardiac Drp1 KO (Kageyama et al EMBO J, 2014), but these Parkin KO mice have little basal phenotype due to germ-line compensation. Here, we assessed the individual and interactive roles of Parkin and Drp1 in adult mouse hearts by conditionally ablating each gene (Cre-Lox at 8 wks), separately and in combination. Parkin KO hearts appeared normal; as reported, Drp1 KO caused lethal cardiomyopathy after 6-7 wks. Cardiac-specific Parkin KO concomitant with Drp1 KO ameliorated the underlying cardiomyopathy by: 1. Increasing survival (94% vs 56%; P<0.0001); 2. Enhancing cardiac contractility (LV FS 36.2±4.0 vs 23.2±1.7%; P=0.01); 3. Decreasing adverse remodeling (LV r/h 5.0±0.5 vs 6.3±0.4; P=0.05); 4. Reducing cardiomyocyte necrosis (1.9±0.5 vs 5.0±1.2%; P=0.05) and replacement fibrosis (33.3±5.3 vs 13.0±1.5%; P=0.02); 5. Attenuating mitochondrial deficiency (5.7±0.4 vs 4.7±0.6 μg/mg; P=0.20). Deleting Parkin did not affect mitochondrial enlargement or respiratory function in Drp1 KO cardiomyocytes, but normalized mitochondrial-associated LC3 and p62, mitophagy markers increased in cardiac Drp1 KO. These studies show how Drp1-mediated mitochondrial fission and Parkin-mediated mitophagy interact to maintain the quality of cardiac mitochondria: Interrupting mitochondrial fission (Drp1 KO) prevents segregation of damaged mitochondrial components into daughter organelles normally targeted for mitophagy; mitophagy thus ultimately consumes fission-defective parent organelles. Parkin KO suppresses generalized mitophagy, postponing (but not preventing) the Drp1 KO cardiomyopathy.
Abstract 92: The Mitochondrial Calcium Uniporter is Necessary for Metabolic Matching of Contractile Demand During Acute Sympathetic Stress
Authors: Luongo Timothy S , Lambert Jonathan P , Yuan Ancai , Zhang Xueqian , Shanmughapriya Santhanam , Gao Erhe , Houser Steven R , Madesh Muniswamy , Elrod John W
Published: July 2021

Contractility is mediated by a variable flux in intracellular calcium (Ca
2+
), which is proposed to be integrated into mitochondria to regulate cardiac energetics. Moreover,
m
Ca
2+
-overload is known to activate the mitochondrial permeability transition pore (MPTP) and induce cell death. Recent studies have reported that the
Mcu
gene encodes the channel-forming portion of the mitochondrial calcium uniporter (MCU) and is required for
m
Ca
2+
uptake. To examine the role of
m
Ca
2+
in the heart, we generated a conditional, cardiac-specific knockout model and deleted
Mcu
in adult mice (
Mcu-cKO
). Loss of
Mcu
protected against myocardial ischemia-reperfusion (IR) injury by preventing the activation of the MPTP. In addition while we found no baseline phenotype,
Mcu
-cKO mice lacked contractile responsiveness to beta-adrenergic receptor stimulation as assessed by invasive hemodynamics (Fig 1) and in parallel were unable to activate mitochondrial dehydrogenases and increase cardiac NADH levels. Further experimental analyses in isolated adult cardiomyocytes confirmed a lack of energetic responsiveness to acute sympathetic stress (isoproterenol failure to mediate an increase in NADH, Fig 2), supporting the hypothesis that the physiological function of the MCU in the heart is to modulate Ca
2+
-dependent metabolism during the ‘fight or flight’ response.
Abstract 97: Epigenetic Signatures Contribute to the Superior Endothelial Cell Identity in Human Induced Pluripotent Stem Cells Derived From Endothelial Cells
Authors: Zhao Mingtao , Hu Shijun , Srinivasan Rajini , Jahaniani Fereshteh , Shao Ning-Yi , Knowles David , Lee Won Hee , Swigut Tomek , Wysocka Joanna , Snyder Michael , Wu Joseph
Published: July 2021

Human induced pluripotent stem cells (iPSCs) can be derived from multiple types of somatic cells by transient overexpression of four Yamanaka factors. Epigenetic memory of the tissue of origin is seen in early passage iPSCs, which may interfere the directed differentiation towards target lineages in disease modeling and drug discovery. Here we derived human iPSC from three types of somatic cells of the same individuals: fibroblast (FB-iPSCs), endothelial cells (EC-iPSCs) and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into endothelial cells by using sequential administration of Activin, BMP4, bFGF and vEGF. EC-iPSCs show higher EC differentiation propensity and EC-specific markers (PECAM1 and NOS3) gene expression in early passage iPSCs than FB-iPSCs and CPC-iPSCs. In vivo, EC-iPSC-ECs display significantly greater revascularization capacity than those of FB-iPSCs and CPC-iPSCs when transplanted to the hindlimb ischemic mice. In addition, transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31+ population and higher EC-specific markers (PECAM1, KDR and ICAM) gene expression by using single cell qPCR. In vitro, EC-iPSC-ECs exhibit better endothelial cell character maintenance along with extensive culturing and passaging. Several chromatin signatures, including H3K27ac, H3K4me1 and p300 were found highly enriched in ECs and EC-iPSCs, but not in human embryonic stem cells (ESCs). Gene ontology analysis indicates that the differentially enriched regions are primarily associated with angiogenesis and vascular development, reflecting the residual epigenetic signatures in EC-iPSCs. Finally EC-specific enhancer markers undergo dynamic changes during the process of EC fate commitment and differentiation, though the majority of them sustain conserved pattern in EC-iPSCs, CPC-iPSCs and FB-iPSCs. In conclusion, these results highlight that the residual epigenetic signatures of tissue of origin may affect lineage differentiation propensity in early-passage human iPSCs.
Abstract 107: RNA Seq Transcriptome Analysis Reveals Genes and Pathways Involved in the Cardiac Protection of VCP Against Pressure Overload
Authors: Leimena Christiana , Chen Xin , Zhou Ning , Stoll Shaunrick , Wang Charles , Qiu Hongyu
Published: July 2021

Aims:
We previously showed that the valosin containing protein (VCP), a member of ATPases associated protein, protects the heart against pressure overload induced cardiac hypertrophy and dysfunction in transgenic (TG) mice. The mechanisms remain unknown. We hypothesize that VCP regulated alteration in transcriptome contributes to its cardio-protection by targeting cardiac cell growth and survival.

Methods and results:
Cardiac-specific VCPTG mice and their litter-matched wild type (WT) mice were subjected to transverse aortic constriction (TAC) for 2 and 5 weeks and compared to the sham mice. By using echocardiography and hemodynamic analysis, cardiac hypertrophy and dysfunction were confirmed in 2 weeks- and 5 weeks- TAC models respectively in WT but not in VCPTG mice. Total RNA was extracted from left ventricular tissues and gene expression was determined by RNA-Seq transcriptome analysis. Upon 2 weeks TAC, 690 differentially expressed genes were identified between VCPTG and WT (Fold Change ≥ 1.5, P value ≤ 0.05). Among these genes, VCPTG TAC mice, compared to WT TAC mice, showed significant activation of the genes linked to transcriptional factors, Protein Kinase CGMP-Dependent Type I (
Prkg1
) and Kruppel Like factor 15 (
Klf15
), the known repressors of cardiac hypertrophy under stress. On the contrary, there is significant increase in cardiac hypertrophy associated genes in WT TAC mice, such as myosin light chain 7 (
Myl7
), periostin (
Postn
) and tropomyosin beta chain (
Tpm2)
, and in fetal gene natriuretic peptide A (
Nppa)
, but these alterations were not observed in VCPTG TAC mice, which may contribute to repression of cardiac hypertrophy in VCPTG mice. In addition, pro-apoptosis genes, such as platelet factor 4 (
Pf4),
Pleckstrin homology like domain A1 (
Phlda1
) and Radical S-Adenosyl Methionine Domain Containing 2 (
Rsad2
), are significantly downregulated continually in 2 weeks through 5 weeks TAC in VCPTG mice, but not in WT TAC, which may contribute to the protection of cell survival in VCPTG mice.

Conclusion:
Significant difference of gene regulation exists between VCPTG and WT in the heart under the pressure overload which may be the mechanism of VCP mediated cardiac protection.
Abstract 108: Exercise Promotes a Cardioprotective Gene Program in Resident Cardiac Fibroblasts
Authors: Lighthouse Janet K , Burke Ryan , Velasquez Lissette S , Dirkx Ronald A , Moravec Christine S , Alexis Jeffrey D , Rosenberg Alex , Small Eric M
Published: July 2021

Exercise and disease both induce hypertrophic cardiac growth, but only disease results in fibrosis and compromised heart function. Transcriptional profiling of resident cardiac fibroblasts (CFs), the primary cellular source of fibrosis, was used to define the gene expression programs (GEP) underlying this divergent functional outcome. Bioinformatic analyses revealed distinct transcriptional responses to exercise and disease, including induction of Rho- and SRF-dependent remodeling genes in disease and NRF2-dependent antioxidant genes in exercise. The expression of a number of antioxidant genes, including metallothioneins (Mt1 and Mt2), are specifically maintained in CFs after exercise and lost in disease. Mice lacking Mt1/2 show signs of cardiac dysfunction after exercise, including cardiac fibrosis, vascular rarefaction, and reduced heart function. Importantly, Mt levels are also reduced in human heart failure (HF) patients, suggesting a potentially conserved cardioprotective role in humans. Non-canonical TGF-β1-mediated p38-MAPK signaling has previously been implicated in HF, therefore we tested the role of p38 signaling in Mt regulation. Pharmacological inhibition of p38 in human HF fibroblasts restores Mt1 and Mt2 expression and blunts the pathological fibroblast phenotype. Taken together, our study defines the transcriptional response of CFs to exercise and disease and reveals a cardioprotective mechanism that is lost in disease.