bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 267: Activation of Non-canonical Estrogen-dependent Pathways to Mitigate Pathological Cardiac Remodeling
Authors: Lopez-Pier Marissa A , Koppinger Matthew , Royal Harris Preston , Strom Joshua , Mayfield Rachel M , Gregorio Carol C , Konhilas John P
Published: March 2022

Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. Mechanisms responsible for loss of CVD protection are unknown. We previously demonstrated that menopause and CVD suppress the AMP-activated protein kinase (AMPK) signaling pathway in mice. We also validated the cellular mechanism by which estrogen (E2) potentiates AMPK activity through a direct interaction of estrogen receptors (ER) with members of the AMPK kinase complex. Because AMPK signaling is down in CVD and menopause, we hypothesized that activation of AMPK will prevent pathological cardiac remodeling in menopausal female mice. First, we demonstrated that E2 potentiates AMPK activity in neonatal rat cardiomyocytes (NRCMs) subjected to energy stress. NRCMs, cultured in estrogen-free media, were treated (10-30 minutes at 100nm) with the electron transport chain uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenyphydrazone (FCCP). As expected, AMPK activity determined by phosphorylation of threonine 172 (p-AMPK172) was increased over controls. Adding 1-100nm of E2 potentiated p-AMPK172 over control-treated NRCMs by 5-fold. Next, we used our novel model of menopause with 4-vinylcyclohexene diepoxide (VCD), which induces gradual ovarian failure, preserving the perimenopause transitional period and androgen secreting capacity of residual ovarian tissue. Starting at 2 months, females received daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days) or sesame oil as vehicle. Peri/menopause were confirmed by vaginal cytology. Menopausal females receiving angiotensin II (Ang II, 800 ng/kg/min via alzet s.c. mini-pump, 14 days) demonstrated exacerbation of hypertension and pathological cardiac remodeling compared to pre- and peri-menopausal mice. Female mice treated with Ang II following surgical removal of ovaries (OVX) experienced a similar exacerbation of cardiac remodeling. Daily adminstration of the AMPK activator (A-769662, s.c. 30mg/kg) prevented pathological remodeling in menopausal and OVX female mice subjected to Ang II. We conclude that AMPK represents a non-canonical target for the mitigation of menopausal susceptibly to CVD.
Abstract 273: Pressure Overload Induces Cardiac Insulin Resistance in an Angiotensin II-Independent Manner
Authors: Zhou Ning , Ye Yong , Shu Hongyang , Fan Jingjing , Zou Yunzeng , Wang Dao Wen
Published: March 2022

Aims:
Pressure overload is able to provoke the cardiac insulin resistance via angiotensin II (AngII). However, the direct effect of pressure overload on the myocardial insulin sensitivity was poorly understood. This study was aimed to explore whether and how pressure overload impairs myocardial insulin sensitivity independent of AngII.

Methods and Results:
To mimic cardiac pressure overload, C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks
in vivo
. Cultured neonatal rat cardiomyocytes (NRCMs) were imposed with mechanical stretch for 24 hours
in vitro
. Although pressure overload dramatically restrained the glucose uptake and expression of glucose transporter 4 (GLUT4) in the myocardial membrane, it didn’t increase the local expression of AngII in TAC mouse hearts and mechanically-stretched NRCMs. C57BL/6 mice were treated with Enalapril and then subjected to TAC for 2 weeks. The glucose uptake and expression of GLUT4 in the myocardial membrane were dramatically restrained in the left ventricles (LVs) of 2 week TAC mice, which were preserved in mice treated with Enalapril. Angiotensinogen knock out (
ATG
-/-
) mice were subjected to TAC for 2 weeks. Angiotensin converting enzyme (ACE)-deficient NRCMs were mechanically stretched. Insulin sensitivity was significantly impaired by TAC in
ATG
-/-
mouse hearts and mechanical stretch in NRCMs evidenced by a notably decreased glucose uptake and expression of GLUT4 in myocardial membrane. Pressure overload impaired the myocardial insulin signals including decreased phosphorylation of insulin receptor, insulin receptor substrate 1 and Akt. Candesartan, an AngII receptor 1 (AT1R) blocker, partly attenuated the myocardial insulin resistance induced by mechanical stress
in vivo
and
in vitro
.

Conclusion:
In conclusion, we unraveled that pressure overload is able to provoke myocardial insulin resistance independent of AngII which was partly mediated by AT1R.
Abstract 287: Metabolic Control of Ischemic Cardiac Injury in Human Cardiomyocytes
Authors: Hu Dongjian , Shah Nikita , Domian Ibrahim J
Published: March 2022

Acute myocardial infarction is a leading cause of cardiac dysfunction and heart failure. As a result, developing in vitro models of myocardial ischemia has been important for the elucidation of the mechanisms of hypoxic injury and the development of novel cardioprotective strategies. During acute ischemia, cardiac injury occurs in minutes to hours in vivo but in hours to days in vitro. This difference in response to ischemia is compounded by differences in cardiomyocytes’ (CMs) metabolism and substrate utilization under standard in vitro growth conditions. An important challenge in modeling ischemia has been to faithfully recapitulate the in vivo cellular response to hypoxic stress. To address this limitation, we have performed comprehensive phenotypic characterization of myocardial metabolism and contractile functions in vitro during normoxia and hypoxia under different metabolic conditions. We show that, unlike CMs in the adult heart, adult murine CMs and hPSC-CMs cultured in glucose as the primary energy source utilize aerobic glycolysis and not oxidative phosphorylation (OXPHOS) for ATP generation. In contrast, CMs cultured with fatty acids as the primary energy source are dependent on OXPHOS for ATP generation and contractile function. Consistent with these findings, only CMs cultured in fatty acid have an acute drop in ATP production and contractility in response to hypoxia. We then show that by modulating the transcriptional activity of hypoxia-inducible factor 1-alpha, its upstream regulator sirtuin, and its downstream target lactate dehydrogenase A, CMs cultured in glucose shift their metabolism from glycolysis to the more metabolically appropriate OXPHOS. Collectively, our results provide novel mechanistic insights into the key regulatory pathways that control hPSC-CMs’ energy metabolism and highlight the significance of comprehensive characterization of CMs’ metabolic and functional responses to ischemic stress in vitro. By creating in vitro models of ischemic injury that can faithfully recapitulate cellular responses of myocardial infarction, it is then possible to identify relevant therapeutic targets for the treatment the disease.
Abstract 292: Novel Synthesis of PFC Nanobubble-magnetic Nanoparticles Conjugate for Acute Myocardial Infarction
Authors: Arokiaraj Mark C
Published: March 2022

Aims:
The potential aim of this study was to prepare this conjugate and analyze its basic characteristics. This could help in myocardial salvage or reduction in infarct size which is potentially very useful.

Methods and Results:
Perfluorocarbon nanobubbles were prepared using standard techniques. PFC nanobubbles-magnetic nanoparticle conjugation was performed and a final solution prepared had a pH of 8.0. The conjugation was performed to form covalent bonding and PBS buffer was used. The final preparation is an emulsion with a PFC content of 25% and 40% concentration in each emulsion of 300 microl. The final volume achieved in the synthesis was 300microl of each concentration, and the final compound was stable. The final sample when evaluated under microscope gives an image shown in figure 1. The polydispersity index was <0.25. The magnetic nanoparticle concentration was 15mg/ml and the particle concentration in 300microl was the particle content was 4.5mg. A drop of emulsion subjected to the magnetic field by a small magnet tends to polarize to one side due to the effect of the magnetic field. These particles are easy and respond quickly to magnetic field. On a glass slide when allowed to dry the conjugate forms a thick film like a mountain, and when a small amount of water is added, and when a small magnet is applied nearby, the particles start moving towards the magnet. The size of the particles is about 100nm by dynamic light scattering.

Conclusion:
Synthesis of magnetic nanoparticle conjugation with PFC nano bubbles is feasible and stable.
Abstract 293: MicroRNA-532 Protects the Heart in Acute Myocardial Infarction by Repressing a Positive Regulator of Endothelial-to-mesenchymal Transition, Prss23
Authors: Bayoumi Ahmed S , Teoh Jian-Peng , Kim Il-man
Published: March 2022

Aims:
Acute myocardial infarction (MI) leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after MI is considered a critical determinant of this process. MicroRNAs (miRs), negative regulators of gene expression, have emerged as important players in MI. We previously showed that miR-532-5p (miR-532) is upregulated by the β-arrestin-biased β-adrenergic receptor antagonist (β-blocker) carvedilol, which activates protective pathways in the heart independent of G protein-mediated second messenger signaling (Figure A-C). Here, we hypothesize that β2-adrenergic receptor/β-arrestin-responsive miR-532 confers cardioprotection against MI.

Methods and Results:
Using cultured cardiac endothelial cell (CEC) and
in vivo
approaches, we show that CECs lacking miR-532 exhibit increased transition to a fibroblast-like phenotype via endothelial-to-mesenchymal transition (EndMT). We also demonstrate that knockdown of miR-532 in mice causes abnormalities in cardiac structure and function as well as reduces CEC proliferation and cardiac vascularization after MI. Mechanistically, cardioprotection elicited by miR-532 is in part attributed to repression of a positive regulator of maladaptive EndMT, prss23 (a vascular protease serine 23) in CECs (Figure D). Lastly, cardiac expression of miR-532 and prss23 is inversely correlated during MI.

Conclusions:
In conclusion, these findings reveal a pivotal role for miR-532-prss23 axis in regulating CEC function after MI, and this novel axis could be suitable for therapeutic intervention in ischemic heart disease.
Abstract 296: Fibroblast Proliferation Dynamics in the Mouse Heart with Myocardial Infarction Injury
in vivo
Authors: Fu Xing , Kanisicak Onur , Khalil Hadi , Molkentin Jeffery
Published: March 2022

After MI, dead cardiomyocytes are permanently replaced by scar tissue mainly composed of fibroblasts and extracellular matrix (ECM) proteins due to the lack of cardiomyocyte regeneration. Previous attempts to characterize fibroblasts after MI were greatly hindered by the lack of specific cardiac fibroblast markers. By employing newly established mouse lines with the Tcf21 locus-driven MerCreMer cDNA or Postn locus-driven MerCreMer cDNA, which when used with a loxP-dependent genetic reporter line will lineage trace either all resident cardiac fibroblasts and all activated cardiac fibroblasts, respectively. Here we show that upon MI injury, cardiac fibroblasts within the area of injury were quickly activated and showed dramatic increases in cellular proliferation followed by an overlapping differentiation phase, resulting in a large number of smooth muscle α-actin (αSMA) positive myofibroblasts within the developing scar region. However, αSMA
+
cardiac fibroblasts were no longer observed by 2 weeks post MI, although collagen deposition increased significantly. Lineage tracing indicates that loss of αSMA
+
fibroblasts within the stabilized scar was due to the loss of αSMA expression in these lineage traced and differentiated fibroblasts, rather than due to their apoptosis. Moreover, additional stimulation of these activated fibroblasts within the stable scar with Angiotensin II - phenylephrine osmotic pumps, which robustly induces new fibroblasts to differentiate and express αSMA in previously uninjured hearts, was not able to re-activate or induce proliferation of the fibroblasts within the scarred region 4 weeks post MI, which suggests that these cardiac fibroblasts were not fully dedifferentiated. We proposed that αSMA
+
myofibroblasts are unique to the early post MI stage in providing structural support before the collagen matrix and additional ECM is fully matured. We will present the results of RNA-sequencing of cardiac fibroblasts isolated from the scar region at different post MI stages to show their more stable intermediate stages.
Abstract 303: C/ebpβ Plays a Crucial Role in Protecting the Heart From Ischemic Injury Through its Phosphorylation at Thr299
Authors: Nah Jihoon , Maejima Yasuhiro , Zhai Peiyong , Nagarajan Narayani , Sadoshima Junichi
Published: March 2022

CCAAT-enhancer-binding protein (C/EBP)β is a basic-leucine zipper transcription factor family member that regulates various cellular mechanisms, including cell differentiation, proliferation, cell death and survival, and inflammation. Post-translational modifications of C/EBPβ, such as phosphorylation, acetylation, and SUMOylation, play an important role in regulating the function of C/EBPβ under stress conditions. However, the function of C/EBPβ in cardiomyocytes is not fully understood. Here, we investigated the role of C/EBPβ in the heart under stress. C/EBPβ is phosphorylated at Thr299 in response to oxidative stress in cardiomyocytes
in vitro
and Mst1, a serine/threonine kinase, mediates this phosphorylation. C/EBPβ is also phosphorylated at Thr299 in the heart during three-hour myocardial ischemia. Overexpression of C/EBPβ and its phosphomimetic mutant (T299E) increases expression of antioxidant-genes, including catalase and MnSOD, and decreases pro-apoptotic genes, such as NOXA, in cardiomyocytes. To elucidate the role of phosphorylation of C/EBPβ at Thr299 in mediating cardiac function during cardiac stress, we generated C/EBPβ-T250E knock-in mice (Thr250 in mice corresponds to Thr299 in humans). C/EBPβ-T250E knock-in mice are born with a normal Mendelian ratio and no apparent basal phenotype. However, similar to the findings in cardiomyocytes, heterozygous C/EBPβ-T250E knock-in mice showed increased expression of anti-oxidant genes, including catalase (1.93±1.0-fold vs. wild-type mice) and MnSOD (5.93±3.24-fold vs. wild-type mice), in the heart at baseline. Moreover, heterozygous C/EBPβ-T250E knock-in mice showed protective effects against three-hour ischemia (% infarct size per AAR: 39.88±3.25 vs. 32.90±2.79). These results indicate that phosphorylation of C/EBPβ at Thr299 plays a crucial role in protecting the heart during ischemic injury by regulating both antioxidant and pro-apoptotic gene expression.
Abstract 305: Cxcr6 Aggravates Myocardial Ischemia/reperfusion Injury Through Inducing Il-17a Producing Nkt Cells
Authors: Wang Shi-Jun , Wu Jian , Zhao Gang , Huang Jia-yuan , Jiang Guoliang , Zou Yunzeng , Ge Junbo
Published: March 2022

Objective:
Immune inflammation is highly associated with myocardial ischemia/reperfusion (I/R) injury. Invariant natural killer T (iNKT) cells are important participants in early-stage innate immune responses. However their roles in I/R-induced cardiac injury are not well defined. We previously showed that chemokine receptor, CXCR6 deficiency prevented heart from I/R injury through reducing cardiac infiltration of inflammatory cells. In this study, we further analyze the role of CXCR6 in activating iNKT cells and iNKT cells-mediated cardiac dysfunction in response to I/R injury.

Approach and Results:
After creation of cardiac I/R injury, the mobilization of iNKT cells in thymus gland, spleen and heart were measured by flow cytometry. Loss of CXCR6 significantly attenuated the cardiac infiltration of iNKT cells, but not affected the recruitment of iNKT cells in thymus gland and spleen. This phenomenon did not change even in the case of intravenous injection with α-galactosylceramide (α-GC), the activator of iNKT cells. Also, Index of Infarct size(IFS)/left ventricular (LV) and IFS/area at risk (AAR), measured by image analyzer after TTC staining, were both lower in CXCR6-/- mice than in wild-type(WT) mice. M-mode echocardiography showed that left ventricular ejection fraction (LVEF) was significantly increased in CXCR6-/- mice when compared with that in WT control mice. In addition, autophagy-related proteins, Beclin-1 and ATG5 were upregulated, and p62 was downregulated in the early stage of reperfusion in CXCR6-/- mice. In vitro, IL-17a secretion was significantly increased in spleen-derived iNKT cells after inducing by α-GC, but α-GC stimulation failed to enhance IL-17a production in CXCR6-/- iNKT cells. Further experiment revealed that I/R-induced protein levels of Beclin-1, ATG5 and p62 were all reversed in CXCR6-/- mice with the pretreatment of recombinant mouse IL-17a, whereas the autophagic flux was improved in WT mice after using anti-IL-17a blocking antibody.

Conclusions:
Our findings highlight CXCR6 as a key chemokine receptor that regulates iNKT cells maturation and cardiac infiltration, which promotes I/R-induced myocardial injury and dysfunction via increasing IL-17a secretion and IL-17a-dependent autophagy inhibition.
Abstract 313: Sphingosine-1-Phosphate and RhoA Signaling Regulate Dynamin-Related Protein 1 and Cardiac Mitochondrial Fission
Authors: Brand Cameron S , Tan Valerie P , Brown Joan H , Miyamoto Shigeki
Published: March 2022

In cardiomyocytes, signaling through RhoA is protective against ischemia/reperfusion injury. Cardioprotective receptor agonists such as sphingosine-1-phosphate (S1P) provide protection through RhoA activation. Mitochondrial fission has been suggested to play a role in cardioprotection, allowing for selective degradation of smaller damaged mitochondria. Our previous studies showed that RhoA signaling can protect cardiomyocytes against ischemia/reperfusion injury by blocking mitochondrial death pathways under oxidative stress, however it is not known if RhoA activation also regulates mitochondrial quality control. We tested the possibility that activated RhoA regulates cardiac mitochondrial fission. Adenoviral expression of constitutively active RhoA in cardiomyocytes caused an increase in small, fragmented mitochondria observed by both fluorescent confocal microscopy and electron microscopy. This mitochondrial fission phenotype was attenuated by inhibition of the downstream RhoA target Rho-associated Protein Kinase (ROCK), or by siRNA knockdown of the primary fission protein Dynamin-related Protein 1 (Drp1). Activated Drp1 causes mitochondrial fission when it translocates from the cytosol to the mitochondria, a process regulated by Drp1 phosphorylation. We determined that expression of active RhoA in cardiomyocytes stimulated phosphorylation of Drp1 at serine-616, and increased mitochondrial levels of Drp1. Both phosphorylation and mitochondrial translocation of Drp1 by active RhoA could be blocked by ROCK inhibition. Endogenous RhoA activation by S1P also increased Drp1 phosphorylation and mitochondrial translocation in a RhoA-dependent and ROCK-dependent mechanism. In conclusion, RhoA activation in cardiomyocytes can increase Drp1 phosphorylation and Drp1 mitochondrial localization, and induce mitochondrial fission. We propose that mitochondrial fission, elicited in response to increased availability of S1P and activation of RhoA in response to ischemia/reperfusion injury, represents a previously unknown pathway for cardioprotection.
Abstract 316: Adap1 Prevents Cardiomyocyte Hypertrophy by Interfering with Integrin Signaling
Authors: Giguere Hugo , Dumont Audrey-Ann , Berthiaume Jonathan , Laberge Gino , Auger-Messier Mannix
Published: March 2022

The Arf GTPase activating protein with dual PH domains 1 (Adap1), a putative regulator of the small G protein ADP-ribosylation factor 6 (Arf6), might be expressed to some extent in the heart. Although Arf6 roles in cardiomyocytes are not well defined, this small G protein was shown to alter surface expression of integrins in other cell types. Considering the importance of integrins on cellular mechanisms of adhesion and spreading,
we hypothesized that Adap1 might impact cardiomyocyte hypertrophy by regulating integrin signaling
. In order to determine the role of Adap1 in cardiomyocytes, we analyzed its cardiac expression in Sprague Dawley rats. Comparatively to its high mRNA expression in the brain, we showed that Adap1 transcript is expressed at relatively low levels in the heart of adult and neonatal rats. However, Western blot detection of Adap1 showed that it is well expressed in the whole heart of adult and neonatal rats at levels reaching 13% and 17% relative to the brain, respectively. Moreover, Adap1 was 3-fold more abundant in isolated rat neonatal ventricular cardiomyocyte (RNVC) comparatively to partially enriched non-cardiomyocyte cells. Adenoviral-mediated overexpression of ADAP1 partially prevented the increase in cell surface area of cultured RNVCs in response to increasing serum concentrations. Interestingly, ADAP1 overexpression completely blocked constitutively active mutant Mek1- and phenylephrine-induced hypertrophy of RNVCs. In these conditions, ADAP1 did not impair the Mek1ca-induced fetal gene program in RNVCs, suggesting that its anti-hypertrophic effect was not mediated by transcriptional regulation. In fact, cell surface biotin labeling indicated that ADAP1 significantly reduced the localization of integrin β1 at the membrane of RNVCs without changing its total expression. Strikingly, integrin β1 was almost completely absent from the surface of RNVCs overexpressing both ADAP1 and Mek1ca. These findings demonstrate for the first time that Adap1 expressed in cardiomyocytes interferes with the surface expression of integrin β1, thereby slowing the development of hypertrophy in vitro. Further in vivo studies are needed to characterize the anti-hypertrophic function of Adap1 in response to pathological stresses.
Abstract 335: Inhibition of Histone Deacetylases Post Myocardial Infarction, Upregulates Wisp-1, a Novel Regulator of Cardiac Angiogenesis
Authors: Wright Lillianne , Herr Daniel , Brown Symone , Kasiganesan Harinath , Menick Donald
Published: March 2022

Introduction:
Re-establishing vasculature after a myocardial infarction (MI) may spare cardiomyocytes from death due to lack of sufficient oxygen. WISP-1 -a secreted matricellular protein that regulates angiogenesis in certain cancers- promotes cell survival in isolated cardiomyocytes
in vitro
. However, the potential role WISP-1 plays post-MI, has not been evaluated. Histone deacetylase inhibition (HDACi) attenuate adverse effects of an MI in small animal models but it is unclear which genes and or targets contribute to this benefit. Our preliminary data shows that
Wisp-1
is upregulated 15-fold in response to MI injury compared to sham-operated mice, but is upregulated 45-fold in mice that are subjected to an MI injury and treated with the HDAC inhibitor, Vorinostat. Therefore, we
hypothesized
that HDACi mediated upregulation of Wisp-1 contributes to beneficial angiogenesis, post-MI
.

Methods:
To test this, we subjected 10-12 week old male mice to ligation of the L.A.D. coronary artery or a sham operation. Mice were injected daily with either DMSO/vehicle, or Vorinostat. Seven days post-MI, mice were euthanized and their heart tissue was assessed for the expression of Wisp-1 and microvasculature. We also assessed the impact of recombinant WISP-1, and respective lentiviral mediated upregulation and shRNA mediated suppression of WISP-1 expression on human coronary artery endothelial cells (HCAECs).

Results:
In vivo
, HDACi mediated upregulation and expression of
Wisp-1
/Wisp-1 is observed at the border zone of infarction and its expression is proximal to microvasculature. Recombinant WISP-1 protein promotes expression of pro-angiogenic genes and phenotypic characteristics in HCAECs. Lastly, lentiviral activation and shRNA-targeted suppression of endogenous WISP-1 respectively enhances and reduces endothelial cell network branching
in vitro
.

Conclusion:
Therapeutic interventions after a heart attack impact the extent of infarct injury, cell survival and overall prognosis. Our studies shown here identify a novel pro-angiogenic target, Wisp-1, that may be useful in post-MI treatment modalities.
Abstract 337: Cytoglobin Promotes Cardiac Stem Cell Survival Against Oxidative Stress via the Upregulation of the NFkB/iNOS Signal Pathway and Nitric Oxide Production
Authors: Zhang Shuning , Li Xiuchun , Jourd’heuil Frances L , Qu Shunlin , Devijian Neil , Bennett Edward , Jourd’heuil David , Cai Chuanxi
Published: March 2022

Cardiac stem cells may serve in regenerative medicine to repair the infarcted heart. However, this approach is severely limited by the poor survival of donor cells. We previously showed that preconditioning human cardiac stem cells (hCSCs) with a nitric oxide (NO) donor enhanced cell survival through activation of survival signaling pathways. Recent studies suggest that the mammalian globin cytoglobin (CYGB) regulates NO metabolism and may regulate cell death. Therefore, the goal of the present study was to test the hypothesis that CYGB may serve important pro-survival functions in hCSCs. We found that CYGB is expressed in hCSCs, in the absence of any significant expression of myoglobin (MB), the primary hemoglobin expressed in adult cardiomyocytes. Through molecular approaches aimed at increasing or decreasing CYGB expression in hCSCs, we found that CYGB functions as a pro-survival factor in response to oxidative stress. This was associated with the upregulation of primary antioxidant systems such as peroxiredoxin 1 (PRDX1) and heme oxygenase-1 and anti-apoptotic factors, including BCL2, BCL-XL, and MCL1. Most significantly, we established that CYGB increased the expression of NFкB-dependent genes including iNOS (NOS2) and that iNOS-dependent NO production was required for a feedforward loop that maintains CYGB expression. Our study delineates for the first time a role for a globin in regulating hCSC survival and establishes novel mechanistic insights in the functional role of CYGB. It provides a rationale for the exploration of the CYGB pathway as a novel molecular target that can be used to enhance the effectiveness of stem cell therapy for ischemic heart disease.
Abstract 339: Hematopoietic-derived Progenitor Cells Identified in Mouse Heart Valves are also Present in Human Mitral Valves
Authors: Hajdu Zoltan , Nagy Mehesz Agnes
Published: March 2022

Tissue engineered heart valve constructs (TEHVCs) are thought to be the solution for the increasing needs of heart valve replacements. Using decellularized xenograft valve matrices reseeded with autologous cells seems to fulfill the demands for durability, lack of coagulation and immune reactions, in the same time allowing growth and adaptability. The presently used cell sources are not viable as the TEHVCs frequently exhibit durability issues due to inefficient seeding and subsequent calcification. The natural valve interstitial cells are very heterogeneous, their role is to maintain the extracellular matrix which is responsible for bearing the mechanical and hemodynamic load. We have previously identified, in murine heart valves, a hematopoietic bone marrow-derived progenitor cell population. These progenitor cells are continuously migrating into the heart valves and appear to participate in the normal homeostasis. The heart valve progenitor cells identified in mice seems a good candidate for TEHVCs, but first, their existence must be validated in human heart valves. We used commercially available, paraffin-embedded, human mitral valve sections from 4 different individuals to immunohistochemically identify the hematopoietic-derived progenitor cells. Combination of anti-CD45 (pan hematopoietic marker) and anti-CD133 (progenitor marker) antibodies revealed a double-positive cell population in the proximal region (toward the annulus), atrial side of the mitral leaflets - exactly where we have found the similar cells in the murine system. By further phenotypic experiments, we have excluded the presence of B- and T-lymphocytes, granulocytes and macrophages. In contrast to the mouse findings, the human CD45
+
/CD133
+
cells are also expressing CD34 which further emphasizes their hematopoietic origin (in mouse the CD34 is not a hematopoietic marker). By co-labeling with CD31 (Pecam-1) markers, we found that the CD45
+
/CD133
+
cells are never part of the endocardium, although they can be observed closely underneath. In conclusion, we have identified the CD45
+
/CD133
+
cells, previously identified in mice as progenitor cells, in human mitral valves. Functional characterization of these cells and their use in TEHVCs is the focus of our future work.
Abstract 341: Disturbance of p53-indcued Long Noncoding RNA Meg3 - FUS Complex by AAV9 System Preserves Heart Function in Myocardial Infarction
Authors: Wu Hongchun , Hao Kaili , Yu You , Han Xinglong , Li Jingjing , Zhao Zhen-Ao , Liu Junwei , Wu Joseph C , Hu Shijun
Published: March 2022

Introduction:
The injured heart undergoes a major process of cellular apoptosis in the initial stage of MI, therefore, the most fundamental method to prevent post-MI remodeling is to suppress cardiomyocyte apoptosis. In this study, we have illustrated the key role of long noncoding RNA,
Maternally expressed gene 3
(
Meg3
), on cardiomyocyte apoptosis and the underlying mechanisms in heart.

Methods:
Neonatal murine cardiomyocytes and human ESC-derived cardiomyocytes were subjected to hypoxia, and cellular apoptosis was evaluated with Annexin V assay. The
Meg3
regulation by p53 was measured by luciferase reporter assay. The complex of
Meg3
and RNA-binding protein FUS (Fused in sarcoma) was determined by EMSA and RIP. Adeno-Associated Virus serotype 9 (AAV9) system was employed to knock down
Meg3
in cardiomyocytes in vivo, and the cardiac function was evaluated by echocardiography and ex-vivo assays.

Results:
We first found that
Meg3
was progressively upregulated in the murine injured heart after MI, and it showed the pro-apoptotic functions in primary cardiomyocytes.
Meg3
could be directly upregulated by p53 during hypoxia condition, and was involved in apoptotic regulation via its direct binding with FUS. The
Meg3
knockdown in cardiomyocytes by AAV9 system could preserve heart function in murine myocardial infarction. Moreover, its pro-apoptotic function was conserved in human cardiomyocytes.

Conclusion:
Together, these results indicate that p53-induced
Meg3
- FUS complex plays an important role on cardiomyocyte apoptosis post-MI, and its specific knockdown in cardiomyocytes with AAV9 system represents a promising method to treat myocardial infarction.
Abstract 344: Cardiac Grafts Engineered From Human Induced Pluripotent Stem Cell Ventricular Pure or Heterogeneous Cardiomyocytes Display Synchronous and Spontaneous Contraction and Reduce Ventricular Tachycardia in Rats with Chronic Heart Failure
Authors: Lancaster Jordan J , Koevary Jennifer , Chinyere Ikeotunye R , Mohran Saffie-Alrahman E , Juneman Elizabeth B , Goldman Steven
Published: March 2022

Background:
Twenty six million people worldwide are diagnosed with chronic heart failure (CHF). With only 5000 heart transplants performed annually, the prognosis for the remaining individuals is poor and new therapeutic options are needed. Previously we have described a cardiac engineered graft comprised of human induced pluripotent stem cell (iPSC) derived cardiomyocytes co-cultured with fibroblasts in a bio-absorbable mesh. Few studies to date have examined comparatively the therapeutic benefits of different cardiac populations. In the present study we engineered grafts generated with either hiPSC derived heterogeneous cardiac myocytes (hetCMs) or ventricular pure myocyte (VMs) populations to compare functional outcomes in a rat model of CHF.

Methods:
Cardiac grafts were generated with hetCMs or VMs (Axiogenesis) by co-culture into a 3D bioabsorbable mesh with human dermal fibroblasts. The hetCMs contain 60% ventricular, 40% atrial and nodal-like cardiomyocytes. The VMs contain 90% ventricular and 10% atrial cardiomyocytes. Grafts were evaluated in culture out to 6 days. Adult male Sprague-Dawley rats underwent permanent left coronary artery ligation and were randomized to Sham, CHF or graft treatment. Hemodynamic pressure measurements were performed at 6 weeks post-ligation with Millar solid state micromanometer pressure catheters. Ventricular tachycardia (VT) induction studies were performed at study endpoint (3 weeks after implant) to evaluate VT susceptibility using methods developed in the laboratory.

Results:
Both HetCM and VM generated cardiac grafts displayed synchronous and spontaneous contractions after 48hrs in culture and maintained an average contraction rate of 67±6 (hetCM) and 71±9 (VM) beats per minute (n=10 per group). Implantation of HetCM and VM grafts lowered (p<0.05) EDP 45% and 14% (n=10) and resulted in decreased susceptibility of VT induction 54% and 65%, respectively (n=4).

Conclusion:
Cardiac grafts engineered with hetCMs or VMs and implanted in a rat CHF model can lower LV EDP in-vivo while decreasing susceptibility of induced VT. This approach raises the possibility that iPSC derived engineered grafts may be used as a treatment for VT in patients with CHF.
Abstract 349: Tracking Human Pluripotent Stem Cell-Derived Cardiomyocytes in Post-Myocardial Infarction Heart with Photoacoustic Imaging
Authors: Qin Xulei , Chen Haodong , Li Kai , Yang Huaxiao , Wu Haodi , Zhao Xin , Daldrup-Link Heike , Wu Joseph C
Published: March 2022

Background:
Molecular imaging has helped unravel potential benefits and barriers of stem cell therapy, yet
in vivo
tracking of cell injection and engraftment with high 3D spatial resolution remains a challenge. This study aims to testify the feasibility of photoacoustic imaging (PAI) as an efficient method to monitor transplanted therapeutic cells in post-myocardial infarction (MI) hearts.

Methods:
Cell penetrating peptide-conjugated photoacoustic nanoparticles (PANPs) were synthesized and applied directly to cultured human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) at the concentration of 4nM for 6h. Post exogenous labeling, cells were injected into mouse MI hearts (n=5) guided by PAI and then monitored by PAI
in vivo
. These engraftments were further confirmed by immunostaining.

Results:
Flow cytometry and confocal imaging indicated that PANP labeling significantly increased the labeling efficiency of hPSC-CMs from 38% to 90% compared to endocytosis labeling. Moreover, calcium imaging and contractile analysis indicated that PANP labeling had no cardiotoxicity to both electrical and mechanical functions of hPSC-CMs. In addition, quantitative polymerase chain reaction (qPCR) showed no adverse effects of PANP labeling on the genes related to cardiomyocyte structures and functions. Finally, animal experiments demonstrated the feasibility of PAI for both injection guidance and followed tracking of hPSC-CMs
in vivo
. The real-time acquired PAI images showed a high spatial resolution (~100 μm) for mice hearts and a high intensity for the labeled cells.

Conclusion:
We utilized PAI for the first time to monitor cardiac regenerative therapy in a preclinical study. PAI demonstrated its superior potential to track cell therapy
in vivo
due to its high spatial resolution, low cost, and non-radioactivity. It will be a useful tool for the investigation of cardiac regenerative therapy.
Abstract 354: Combination of Triiodothyronine and Dexamethasone with Flexible Matrigel Substrate Improves Structural and Electrophysiological Maturation of Human iPSC-derived Cardiomyocytes
Authors: Wang Lili , Gomez-hurtado Maria de las nieves , Blackwell Daniel , Kim Kyungsoo , Parikh Shan , Knollmann Bjorn C
Published: March 2022

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have promise for disease modeling and cell therapy, but electrophysiological immaturity (ie. large pacemaker currents, small I
K1
) and structural immaturity (ie. lack of t-tubules) remain major limitations. Here, we test whether the combination of triiodothyronine (T
3
) and dexamethasone (Dex) with a soft Matrigel substrate improves hiPSC-CMs maturity. T
3
& Dex or vehicle were added to cardiac induction media for 14 days. Day 30 hiPSC-CMs were plated either on hard or soft Matrigel-coated substrate for 5 days and studied using confocal imaging and voltage clamp. T
3
& Dex treatment significantly increased the width and volume of hiPSC-CMs cultured on soft but not on hard substrate (
Fig.1
). T
3
& Dex treated hiPSC-CMs exhibited organized subcellular Ca release (
Fig.2
) and evidence of t-tubule development, possibly as a result of increased Bin-1 and Caveolin-3 membrane trafficking. The combination of T
3
& Dex and soft substrate increased Ca spark frequency and the amount of Ca released per spark (
Fig.3
). T
3
& Dex treatment significantly accelerated Ca transient decay under field stimulation and increased sarcoplasmic reticulum Ca-ATPase activity. Compared to vehicle, T
3
& Dex treatment significantly improved electrophysiological maturity: Pacemaker current (I
f
) density was 3-fold reduced (-2.7±0.35
vs.
-6.2±1.3 pA/pF) and inward rectifier current (I
K1
) density was 4-fold increased (-19.3±2.2
vs.
-4.4±0.5 pA/pF). Combining T
3
& Dex and soft Matrigel substrate enhances structural and functional maturation of hiPSC-CMs, which will increase the utility of hiPSC-CM for disease modeling and cell therapy.
Abstract 370: Wt1 Influences Regenerative Angiogenic Function of the Vascular Endothelium
Authors: Ogley Robert , McGregor Richard , Hastie Nick , Hadoke Patrick
Published: March 2022

Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis (new blood vessel formation from existing vasculature) fails to consistently induce sufficient reperfusion to facilitate repair, a targeted approach to manipulating angiogenesis is clinically desirable. The Wilms’ tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that Wt1 influences the angiogenic function of endothelial cells.

Wt1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from Wt1-GFP reporter mice. Inducible deletion of Wt-1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p<0.05 relative to
Cre
and vehicle controls, n=5-8).

In vivo
, Wt-1 expression was evident in vascular endothelial cells and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia. Less expression was detected by day 28. VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density (histology, immunohistochemistry) or expression of angiogenic factors (qRT-PCR) in sponges inserted beneath the skin of mice (20 days). To further understand the role of Wt1 in angiogenesis, transcriptomic analysis was performed for RNA expression in Wt1
+
and Wt1
-
cells isolated (FACs) from sponges 7 and 21 days after implantation in Wt1-GFP mice. Wt1
+
cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including vasculature development (D7: p=10
-7.43
, D21: p=10
-3.47
, n=3) and angiogenesis (D7: p=10
-3.90
, D21: p=10
-4.74
, n=3).

These results show that deletion of endothelial Wt-1 delays, rather than permanently inhibiting, angiogenesis: suggesting a more complex mechanism of action than reported previously. Transcriptomic data suggest that expression in endothelial cells (and other cells in the endothelial lineage) contributes to tissue repair in ischaemia. This work has improved our understanding of the regulatory role of Wt1 in angiogenesis.
Abstract 371: Assessment of Catheter Position Above or Below the Aortic Valve by Evaluation of Characteristics of the Local Electro Gram: an Acute Canine Study
Authors: Pa Deepak , Witt Chance , Sugrue Alan M , Suddendorf Scott , Powers Joanne , Ladewig Dejae , Drakulic Budimir , Foxall Thomas , Asirvatham Samuel
Published: March 2022

Background:
Ablation in the sinuses of Valsalva (SoV) within the aorta is fraught with risk, owing to the proximity of the coronary arteries. Assessment of catheter tip position is not very easy at this location owing to complex local anatomy and poor images from available modalities.

Objectives:
We propose the use of novel local unipolar electrogram characteristics using a new signal recording system to guide the assessment of catheter tip position in this region.

Methods:
We recorded unipolar signals from above the SoV, within the coronaries and below the SoV using the PURE-EP™ (BioSig Technologies, MN) signal processing system in 4 anesthetized canines in the setting of an acute study. We used fluoroscopy, intracardiac echocardiography and angiography for confirmation of the position of the catheter tip. We recorded changes in the signals when transitioning between (1) the coronary arteries and the SoV and (2) the SoV and below the SoV.

Results:
The unipolar signal showed a negative current of injury (COI) when the catheter tip was present in the SoV, whereas within the coronary artery or below the SoV, the COI was positive. We propose that the difference in the distribution of ventricular muscle below and above the SoV is responsible for this phenomenon. These changes in the COI were noted immediately when transitioning from one location to another and were present reproducibly in all the dogs. (
Figure 1
)

Conclusions:
Analysis of the changes on the local electrogram can provide information about the location of the catheter tip which could prove critical in saving lives.
Abstract 373: Effects of Coronary Wall Mechanics on Smooth Muscle Cell Phenotypic Switch and CD44
+
Mesenchymal Cell Repopulation in Saphenous Vein Grafts
Authors: Ruiter Matthijs S , Piola Marco , Garoffolo Gloria , Gianazza Erica , Agrifoglio Marco , Saccu Claudio , Zoli Stefano , Zanobini Marco , Banfi Cristina , Soncini Monica , Fiore Gianfranco B , Pesce Maurizio
Published: March 2022

Background:
Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for bypass grafting, especially in multi-vessel coronary artery disease. Early remodeling induced by altered wall mechanics has been recognized to play a key role in SV graft disease. The mechanism remains, however, unknown.

Aim:
To investigate mechanical factors involved in early graft remodeling, we characterized SV-derived smooth muscle cells (SMCs) after both ex vivo coronary-like mechanical stimulation of SV segments and in vitro unidirectional strain.

Methods:
SV segments from patients receiving coronary artery bypass grafts were stimulated in a custom-made coronary pulse-duplicator bioreactor. After 7 (n=6) or 14 (n=5) days, stretched and control SVs were fixed and stained for immunofluorescence. Additionally, SMCs isolated from SVs of 7 patients undergoing saphenectomy were subjected to uniaxial cyclic strain (10% elongation, 1 Hz) for 24 or 72 hours using a Flexcell platform. SMCs analysis was performed by western blotting and mass spectrometry-based secretome analysis.

Results:
Coronary stimulation elevated apoptosis of SV medial cells after 7 days, and consistently reduced the percentage of cells positive for contractile markers α-SMA and calponin. Conversely, synthetic phenotype marker tropomyosin-4 (TM4) and early contractile marker SM22α were elevated at T14. Mesenchymal marker CD44 was markedly upregulated in cells populating the media after 14 days of stimulation. In accordance, strained SMCs displayed decreased α-SMA and SM22α, and increased TM4 protein expression after 72h. Analysis of the supernatant showed a significant increase of plasminogen activator inhibitor-1 and thrombospondin 1.

Conclusions:
Mechanical stimulation of SVs leads to apoptosis of medial cells and a decrease of contractile SMC markers, followed by repopulation with cells expressing the mesenchymal marker CD44. In addition, unidirectional strain induces a switch of SMC phenotype and secretion of proteins related to vascular remodeling. We are currently investigating whether CD44
+
cells derive from SMCs undergoing phenotypic switch, or from progenitor cells localized in the adventitia.
Abstract 387: Weighted Gene Co-Expression Network Analysis Identification and Experimental Validation of Genetic Determinants of Right Ventricular Function
Authors: Rau Christoph , Lee Katie , Chukwuneke Jeffrey , Liu Yanan , Li Qing , Wang Yibin , Tsai Emily J
Published: March 2022

Right ventricular dysfunction (RVD) portends worse outcomes in chronic heart failure (HF), irrespective of left ventricular function. Despite the significance and prevalence of RVD, its molecular pathobiology is poorly understood. Gene networks can identify and explore complex relationships between genes, gene pathways, and phenotypes. We used weighted gene co-expression network analysis (WGNA) of human heart samples to identify genetic determinants of RV function and validated candidate genes in animal models of RV versus LV dysfunction.

We isolated and sequenced total RNA from explanted left and right ventricles of ischemic cardiomyopathy (ICM) patients (n=10) who underwent cardiac transplantation. Non-failing donor hearts that were declined for transplant were used as controls (n=5). Half the ICM patients had severe RV failure (RVF) based upon hemodynamic indices (mean±SEM) with mean RA 25.8±1.7 mmHg, RA:PCWP ratio 1.05±0.11, and PA pulsatility index 1.6±0.2.

Using direct gene membership comparison and z-score analysis, we found that more than half the modules in RV-specific networks are shared by combined RV-LV gene networks. We used principle component analysis to examine modules that correlated with RVF-related phenotypes. Two modules unique to the RV network identified selenium-binding protein gene
Selenbp1
as a potential driver of RVF. We also identified a strong association between anti-apoptotic gene
Mif
and vesicle fusion gene
Snap47
with RA:PCWP and PA pulsatility indices, suggesting potential important roles of these genes in RVF. Finally, peroxiredoxin 5
PRDX5
and E3 ubiquitin-protein ligase
SHPRH
were associated with protection against RVF. We sought to validate these candidate gene drivers of RV function in mouse models of RV versus LV-specific dysfunction induced by pulmonary artery banding (PAB) and transverse aortic constriction (TAC), respectively.
PRDX5
expression was markedly reduced in RV of PAB versus Sham but unchanged in LV of TAC versus Sham.
Selenbp1
was diminished in both RV of PAB and LV of TAC mice, relative to Sham. Expression of other candidate genes did not vary significantly with PAB or TAC. Our preliminary findings suggest that cardiac
PRDX5
may play an important role in regulating RV function.