bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 168: Contribution of Vascular Endothelial Growth Factor Signaling to Fate Specification in Cardiomyocytes
Authors: Lu Taylor Y , Domigan Courtney K , Antanesian Vaspour , Nakashima Yasuhiro , Nakano Atsushi , Iruela-Arispe Luisa
Published: July 2021

Vascular endothelial growth factor (VEGF) is one of the pivotal proangiogenic growth factors that has long contributed to our knowledge of blood vessel and circulatory maintenance as well as angiogenesis in both pathology and pathophysiology. However, the non-canonical functions of VEGF in cardiac morphogenesis have not been well characterized. Here, we examined how VEGF regulates cardiomyocyte cell fate.

Using chimeric embryos harboring both wild type and VEGF-null embryonic stem cells, we observed that derivatives of VEGF null cells were preferentially recruited to the atrium of the heart in comparison to the ventricles. To further provide physiologic context of this finding, we used reporter-LacZ staining and RT-PCR and found that endogenous VEGF was indeed expressed at much lower levels in the atrium but highly expressed in the ventricle early in cardiac morphogenesis. These data lead to our hypothesis that cell-autonomous expression of VEGF is a determinant of atrial vs. ventricular cardiomyocyte cell fate. To test this hypothesis, we used a VEGF knock-in mouse model of Sm22Cre x Rosa 26 VEGF. VEGF overexpression in cardiomyocytes (and smooth muscle) at E8.5 resulted in lethality by P1 and thickened atrial and ventricular walls in mutant embryos as characterized by histology (H&E, IF). We further explored the molecular changes underlying this phenotype via microarray and RT-PCR and find disruptions in molecular markers necessary for wall development, specifically: Notch-1, BMP10, Nrg-1.

Taken together, our data indicates that aberrant embryonic VEGF signaling disrupts several critical signaling pathways and that overexpression leads to disruption of cardiomyocyte proliferation and cardiac morphogenesis. These findings add to the foundation of better understanding heart development, laying the groundwork for future therapy of congenital and acquired cardiac disease.
Abstract 174: Role of Cardiomyocyte-Localized Nuclear Factor (NF)-κB in Diet-Induced Diabetic Cardiomyopathy
Authors: Goel Mehak , Ismahil Mohamed A , Hamid Tariq , Prabhu Sumanth D
Published: July 2021

Role of Inflammatory signaling in the development of obesity- and type 2 diabetes (T2D)-associated cardiomyopathy remains unclear. We hypothesized that cardiomyocyte-specific inflammatory activation, independent of systemic inflammation, is sufficient for the genesis of diabetic cardiomyopathy. Wild-type (WT) and transgenic (Tg) C57BL/6 mice with myocyte-restricted overexpression of phosphorylation-resistant IκBα (IκBαS32A,S36A) were fed a high fat (HFD, 45% kcal fat) or control diet (CD, 10% kcal fat) for 28 weeks to induce obesity and T2D. These Tg mice exhibit attenuated NF-kB activation, a central regulator of inflammation. After 28 weeks of feeding, WT HFD mice exhibited increased body weight, hyperglycemia, hyperinsulinemia, insulin resistance, and glucose intolerance. In contrast, although HFD-fed Tg mice exhibited significantly (p < 0.05) increased body weight, the mice still maintained normal fasting blood glucose, insulin levels, and insulin sensitivity compared to CD Tg mice, indicating absence of overt T2D. Echocardiography revealed that both HFD-fed WT and Tg mice developed concentric left ventricular (LV) hypertrophy with increased septal and posterior wall thickness, decreased LV end-diastolic diameter and had unchanged LV ejection fraction suggesting development of LV hypertrophy in both strains regardless of the severity of the metabolic disorder. Analysis of mononuclear phagocyte cell profiles revealed similar trends in WT and IκB Tg mice. HFD fed mice had higher levels of pro-inflammatory (CD11b+Ly6chi) and anti-inflammatory (CD11b+Ly6clo) monocytes in the blood, heart, spleen and adipose tissue compared to CD fed mice. Analysis of antigen presenting cells indicated that HFD fed mice in both genotypes had significantly increased levels of both classical (CD11c+CD45R–) and plasmacytoid (CD11cintCD45R+) dendritic cells in blood, heart, spleen and adipose tissues compared to respective CD fed mice. Thus, we conclude that cardiomyocyte inflammation is central to the development of metabolic abnormalities related to obesity, whereas neither cardiomyocyte-specific nor systemic insulin resistance is required for the development of the hypertrophic cardiomyopathic phenotype.
Abstract 175: Genetic Ablation Of S-nitrosoglutathione Reductase In Mice Enhances Proliferative Expansion Of Adult Heart Progenitors And Myocytes Post Myocardial Infarction
Authors: Hatzistergos Konstantinos E , Paulino Ellena C , Dulce Raul A , Takeuchi Lauro M , Kulandavelu Shathiyah , Balkan Wayne , Kanashiro-Takeuchi Rosemeire M , Hare Joshua M
Published: July 2021

Objectives:
The molecular pathways underlying the proliferative activity of adult cardiac myocytes and stem/progenitors in response to heart damage remain elusive. Since perturbing nitroso-redox balance in mice by genetic deletion of GSNOR (GSNOR
-/-
) confers resilience to experimental myocardial infarction (MI), we investigated whether GSNOR knockout influences the proliferative activity of the post-MI heart.

Hypothesis:
Knockout of GSNOR in mice enhances the proliferative expansion of CMs and cKit
+
cardiac stem cells (CSCs) in response to MI.

Methods:
Wild-type (WT) and GSNOR
-/-
mice (n=5/ group) underwent experimental MI. To assess proliferative activity, animals received intraperitoneal injections of 5-bromodeoxyuridine (BrdU) at selected time-points during the first 2 weeks post-MI. Immunnohistochemical evaluation was performed 1 month post-MI.

Results:
Confocal immunofluorescence revealed that GSNOR
-/-
hearts exhibited higher rates of BrdU incorporation in CSCs after MI (10.9%±4.99% of WT CSCs compared to 15.9%±3% of GSNOR
-/-
CSCs,
p
=0.02). Similarly, there were ~3-fold more BrdU
+
/Tropomyosin
+
cardiomyocytes in the infarct zone of GSNOR
-/-
mice compared to WT (
p
<0.05). Immunohistochemical evaluation of cardiac troponin-T
+
cardiomyocytes co-expressing the mitotic marker ser-10 phosphorylated histone H3 (H3P) showed further that cardiomyocyte mitosis was 2.4-fold greater in GSNOR
-/-
compared to WT mice (
p
<0.05), whereas the presence of aurora-b kinase in the cleavage furrow of GSNOR
-/-
cardiomyocytes substantiated their competence for mitosis after MI. The rate of cardiomyocyte apoptosis after MI, was not different between GSNOR
-/-
and WT mice, as shown by activated cleaved caspase-3 immunofluorescence.

Conclusions:
Collectively, our findings suggest that protein S-nitrosothiol turnover by GSNOR regulates proliferation of cardiomyocytes and CSCs in the adult heart in response to damage. These findings have therapeutic implications for the treatment of heart disease since they reveal novel pathways by which nitroso-redox balance influences cardiac repair.
Abstract 182: Mimetic peptide overcomes dysregulated L-Type Calcium Channel density and recovers myocardial function
Authors: Rusconi Francesca , Miragoli Michele , Di Pasquale Elisa , Rocchetti Marcella , Ceriotti Paola , Carullo Pierluigi , Caprari Silvia , Viggiani Giacomo , Cazade Magali , Chemin Jean , Bang Marie-Louise , Polticelli Fabio , Zaza Antonio , Condorelli Gianluigi , Catalucci Daniele
Published: July 2021

Voltage dependent L-Type calcium-channels (LTCCs) are located on the cardiomyocyte membrane and regulate cardiac contraction and rhythmicity. In human pathologies, such as heart failure (HF), decreased inward calcium current (I
Ca
) is frequently observed. Here, we generated a mimetic peptide (MP) that targets LTCCs and restores impaired intracellular calcium homeostasis through a novel mechanism. Effective delivery of MP, fused with a cell penetrating peptide, was found to correct Ca2+ alterations in a mouse model of HF, in human cardiomyocytes derived from induced pluripotent stem-cells. These data provide a proof-of-concept supporting a therapeutic role for MP to treat human diseases related to LTCC abnormalities.

Category: heart failure biology
Abstract 191: Ras GTPase-activating Protein SH3 Domain Binding Protein 1 (G3BP1) Regulates The Induction Of Stress Granules During Cardiac Hypertrophy
Authors: Sayed Danish
Published: July 2021

Stress granules (SGs) are dynamic, microscopically visible, cytoplasmic bodies that play a major role in mRNA metabolism (e.g. sorting, storage, decay) and induced in cells during stress conditions like starvation, oxidative strain or growth. With substantial role in cancer and neurodegenerative diseases, these granules have never been studied during cardiac hypertrophy, or in the heart in general. Several studies have identified independent proteins, mostly mRNA binding proteins that are part of these granules, some of which are sufficient to nucleate the assembly in quiescent cells even without stress. One such mRNA binding protein is Ras GTPase-activating protein SH3 domain binding protein 1 (G3BP1), which increases during cardiac hypertrophy via posttranscriptional regulation. Thus, we hypothesized that G3BP1 might be involved in the induction of SGs during hypertrophy and hence in regulating mRNA processing and gene expression. Our aim was to investigate, 1) if these SGs appear in hypertrophied hearts and 2) if G3BP1 is necessary and sufficient to induce them during hypertrophic stimuli. In vivo staining of TIA-1/TIAR (SG marker) in mouse hearts subjected to sham or transaortic coarctation (TAC) surgeries showed accumulation of these granules with cardiac hypertrophy. Similar induction was seen in isolated, cultured, rat neonatal cardiac myocytes with hypertrophic stimulation (Endothelin1) or overexpression of G3BP1 alone (>60% of myocytes stained for SG). Conversely, switch to growth-inhibited conditions or knockdown of G3BP1 in hypertrophying myocytes was sufficient to prevent the assembly of these structures. Co-staining with other components of these granules like TIA-1/TIAR or proteins specific to P bodies, like decapping enzyme 1 validated these structures as SGs in cardiac myocytes. Interestingly, a long non-coding RNA, Gas5 (Growth Arrest Specific 5) that is validated binding partner of G3BP1 sequestered to perinuclear focal locations in myocytes stimulated with ET1, suggesting growth-induced recruitment to SGs. While we are still in process of examining G3BP1 targets that are recruited to SGs and their role in hypertrophy development, we have concluded that G3BP1 is required for the induction of SGs during cardiac hypertrophy
Abstract 193: 18-HEPE, An EPA Metabolite Released By Macrophages, Prevents Against Pressure Overload-induced Cardiac Remodeling
Authors: Endo Jin , Sano Motoaki , Fukuda Keiichi , Arita Makoto
Published: July 2021

N-3 polyunsaturated fatty acids (PUFAs) have potential cardiovascular benefit, although the mechanisms underlying this effect remain poorly understood. The present study investigated the impact of the enrichment of n-3 PUFAs in the setting of pressure overload induced cardiac hypertrophy using fat-1 mouse, a genetic model with elevated n-3 PUFA levels in tissues, as well as the possible cellular mechanism involved in such a role.

The fat-1 mice subjected to pressure overload by transverse aortic constriction showed less infiltration of macrophages and decreased interstitial fibrosis, leading to the preserved cardiac function as compared to wild type (WT) mice. Bone marrow (BM) transplantation experiments revealed that fat-1 transgenic BM cells, but not fat-1 transgenic cardiac cells, contributed the anti-remodeling effect, which was comparable between fat-1 and WT mice transplanted with fat-1 transgenic BM, but was absent in fat-1 mice transplanted with WT BM. In the co-culture assay with cardiac fibroblasts, the fat-1 transgenic macrophages showed anti-inflammatory and anti-fibrotic activities. Lipidomic analysis revealed selective enrichment of eicosapentaenoic acid (EPA) in fat-1 transgenic bone marrow (BM) cells and an EPA-metabolite 18-hydroxyeicosapentaenoic acid (18-HEPE) in fat-1 transgenic macrophages. 18-HEPE-rich milieu in the fat-1 transgenic heart was generated by BM-derived cells, most likely macrophages. 18-HEPE inhibited macrophage-mediated proinflammatory activation of cardiac fibroblasts in culture, and in vivo administration of 18-HEPE significantly attenuated the inflammation and fibrosis of hypertrophied hearts, resulting in improvement of cardiac function.

We found that the elevated n-3 PUFA level in BM-derived macrophages is responsible for cardiac protection, and 18-HEPE exhibited cardioprotective potential when administered in vivo.
Abstract 195: RhoA Controls Myofibroblast Characteristics
Authors: Jatho Aline , Kittana Naim , Schenk Kerstin , Ramba Beate , Zimmermann Wolfram-Hubertus , Lutz Susanne
Published: July 2021

Purpose:
RhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblastic phenotype showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton, a process known to be strongly regulated by RhoA.

Methods:
To investigate the role of RhoA in neonatal rat cardiac fibroblasts we used a lentivirus-based knockdown (RhoA-KD-CF). In addition, wild type (wt) CF were treated with 10 μM Fasudil for ROCK inhibition or 5 μg/mL Tubastatin A (Tub A) for tubulin-specific deacetylase HDAC6 inhibition. Cytoskeletal proteins were analyzed by immunoblot and immunofluorescence. Adhesion velocity and migration was determined by microscopy and the serum-driven proliferation rate by nuclei counting.

Results:
Compared to control cells (shControl) RhoA-KD-CF develop an epithelial-like morphology lacking stress fibers and higher order actin structures like geodesic domes. The orientation of focal adhesions sites along the cell stress axis was also impaired. This phenotype could be mimicked by the treatment of CF with Fasudil. Furthermore, in RhoA-KD-CF cytoskeletal proteins were found to be unchanged except for a decrease in the myofibroblast marker smooth muscle actin by 43% and an increase in acetylated tubulin by 57% without a change in the expression of HDAC6. In order to analyze the impact of both changes we investigated the migration and proliferation rate of CF. First, the reduction of RhoA accelerated the adhesion but decelerated migration (shControl 4043±316 nm/h versus shRhoA 3021±153 nm/h) and second, the serum-driven proliferation rate of RhoA-KD-CF was reduced by 50%. Interestingly, treatment of wt-CF with Fasudil significantly decreased migration velocity by 62% but had no effect on the proliferation rate, whereas Tub A only slightly decreased migration velocity but reduced proliferation rate by 60%.

Conclusion:
RhoA influences the two main myofibroblast characteristics, migration and proliferation, by interfering with the actin and tubulin cytoskeleton via ROCK and HDAC6, respectively.
Abstract 2: Automated Dissociation Of Neonatal Hearts And Immunomagnetic Purification Of Cardiomyocytes, Cardiac Fibroblasts And Endothelial Cells
Authors: Kernbach Manuel , Riesen Josephine , Wiencierz Anne Maria , Bosio Andreas , Christalla Peter , Eckardt Dominik
Published: July 2021

Pure cardiovascular cell lineages are a requisite for an unbiased and cell-type specific investigation of (patho-) physiological mechanisms involved in heart failure. Moreover, pure heart cells are needed as building blocks for cardiac tissue engineering. Therefore, we developed a rapid and automated heart dissociation protocol followed by an immunomagnetic enrichment of the three major heart cell types, namely cardiomyocytes (CMs), cardiac fibroblasts (Fibs) and endothelial cells (Endos) from both, mouse and rat neonatal hearts.

First, we developed an enzyme mix optimal for the dissociation of neonatal mouse and rat hearts by screening our enzyme library. Next, we optimized the dissociation by utilizing our gentleMACS technology resulting in a fast (1 h), robust, and fully automated heart dissociation protocol. Analysis of the dissociated heart cells prior to cell purification showed: (i) high cell vitalities (>90%), (ii) high frequencies of α-actinin-positive CMs (ca. 60%) and (iii) vimentin-positive non-CMs with a frequency of ca. 40%. In order to selectively enrich various cell-types from these heterogenous cell populations, we performed a cell-surface marker screen. We identified several candidates for the composition of antibody cocktails enabling selective enrichment of mouse and rat CMs, Fibs or Endos with purities of up to 97%. CMs grown in 2D cultures showed spontaneous beating activity and the expression of sarcomeric proteins. Additionally, heart cells were cultured on decellularized mouse heart slices. Analysis of the slice cultures after recellularization showed (i) partial restoration of synchronous contractions and (ii) orientated and elongated heart cells. Purified Fibs were highly proliferative and could be expanded 34-fold over an analyzed culture period of 15 days. Finally, functionality of the purified endothelial cells was proven by Dil-LDL endocytosis.

In summary, we established an automated protocol for the dissociation of neonatal hearts, enabling the subsequent immunomagnetic enrichment of CMs, Fibs and Endos which can readily be utilized for 2D cell culture assays or to generate in vitro heart muscle models and surrogate tissue for myocardial repair.
Abstract 201: MED13 Dependent Signaling From The Heart Confers Leanness By Enhancing Metabolism In Adipose Tissue And Liver
Authors: Baskin Kedryn K , Grueter Chad E , Kusminski Christine M , Holland William L , Satapati Santhosh , Burgess Shawn C , Scherer Philipp E , Newgard Christopher B , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

The heart requires a continuous supply of energy but has little capacity for energy storage and thus relies on exogenous metabolic sources. We previously demonstrated that cardiac MED13 modulates systemic energy homeostasis in mice. In the present study, we sought to define the extra-cardiac tissue(s) that respond to cardiac MED13 signaling. Cardiac over-expression of MED13 in mice (MED13cTg) confers a lean phenotype that is associated with increased lipid uptake, beta-oxidation and mitochondrial content in white adipose tissue (WAT) and liver. Cardiac expression of MED13 decreases metabolic gene expression and metabolite levels in heart and liver but enhances them in WAT. Although exhibiting increased energy expenditure in the fed state, MED13cTg mice are metabolically flexible and adapt to fasting. These findings demonstrate that MED13 acts within the heart to promote systemic energy expenditure in extra-cardiac energy depots and point to an unexplored metabolic communication system between the heart and other tissues.
Abstract 202: Dysregulation of the Small Organic Carnitine Transporter and Carnitine in Heart Failure
Authors: Bedi Kenneth C , Snyder Nathaniel W , Brandimarto Jeff , Mesaros Clementina , Blair Ian A , Margulies Kenneth B , Rame J. E
Published: July 2021

Introduction:
Advanced heart failure (HF) is characterized by metabolic abnormalities. We have recently shown myocardial lipid species to be significantly decreased in non-diabetic HF. The finding of deficient carnitine species has directed us to examine a key gene SLC22A5 the high-affinity carnitine transporter. Known mutations of this gene lead to primary carnitine deficiency and cardiomyopathy.

Methods:
Left ventricular samples procured at the time of orthotopic heart transplantation (OHT) from non-diabetic idiopathic dilated cardiomyopathy (DCM) n=16, and brain-dead organ donors without a history of diabetes or HF (NF) n=18 formed the study sample. All hearts received in situ cold cardioplegia. Lipids including carnitines were quantitated with liquid chromatography and high-resolution mass spectrometry (LC-HRMS) following the Folch extraction method. mRNA expression data of target genes including SLC22A5 from a separate study of n=117 DCM patients and n=67 NF patients p≤0.05 were analyzed.

Results:
Representative trends for all carnitine species assayed were shown to be significantly reduced in DCM. The figure below represents the mRNA expression between DCM and NF patients. There was a statistically significant reduction of SLC22A5 expression in the DCM population (Figure: ***p≤ 0.0001, dots 95
th
percentiles).

Conclusions:
A statistically significant deficiency of carnitine species and a reduction in the expression of the carnitine transporter SLC22A5 was demonstrated in non-diabetic HF. Future studies will attempt to resolve the mechanisms behind these observations.
Abstract 21: Quantification of Total Myocyte Regeneration Following Intracoronary Cardiosphere-Derived Cell (icCDC) Treatment in Swine with Chronic Myocardial Ischemia
Authors: Weil Brian R , Ribbeck Amanda , Suzuki Gen , Leiker Merced M , Fallavollita James A , Canty John M
Published: July 2021

Objective:
icCDCs improve function of ischemic myocardium and increase cardiomyocyte nuclear density but the extent to which this is explained by an increase in nuclei per cell vs. the formation of new myocytes is unclear. We aimed to address this issue and quantify new myocyte formation throughout the left ventricle (LV) after global icCDC treatment in swine with hibernating myocardium.

Methods:
Swine with a chronic LAD stenosis (N=29) received either ~35 x 10
6
icCDCs or no treatment. Regional function (echocardiography) and myocyte morphometric indices were assessed 1 month later. Transverse and longitudinal tissue sections were used to measure regional myocyte nuclear density, diameter, length, and nuclei/cell, from which myocyte volume and myocytes per gram of myocardium were calculated.

Results:
icCDC-mediated improvements in regional function were accompanied by an increase in myocyte nuclear density without a change in the number of nuclei/cell (Table). Compared with untreated animals, icCDC-treated animals exhibited a reduction in myocyte volume and a reciprocal increase in the number of myocytes per gram of tissue. Anatomic LV hypertrophy did not occur, however, as LV mass/body mass ratio was not different between groups (untreated: 2.5 ± 0.1 vs. icCDCs: 2.3 ± 0.1).

Conclusion:
These data demonstrate that icCDCs produce significant myocyte regeneration throughout the regionally ischemic heart that is dissociated from alterations in LV mass. Changes in myocyte nuclear density do not reflect an altered number of nuclei/cell and quantitative estimates suggest that intracoronary delivery of CDCs to the entire LV increases the number of myocytes by ~25% in just 4 weeks.
Abstract 211: Automated and Non-invasive Estimation of QT Interval from Microscopy Images of Stem Cell-Derived Cardiomyocytes
Authors: Maddah Mahnaz , Loewke Kevin
Published: July 2021

A promising application of induced pluripotent stem cells (iPSCs) is the generation of patient-specific cardiomyocytes (CMs), which can be used for drug development and safety testing related to cardiovascular health. iPSC-derived CMs can be used for preclinical testing of new drugs that may cause drug-induced arrhythmia or long QT syndrome, as well as post-market safety testing of existing drugs. The measurement of QT interval for iPSC-derived CMs is commonly analyzed using electrophysiological potentials captured by a micro-electrode array (MEA). While such systems are the current standard for characterization, they can be expensive and low-throughput, require high cell plating density, and due to the direct contact between cells and electrodes, may cause undesirable cellular response.

Here, we present a new method to non-invasively measure the QT-interval in iPSC-derived CMs using video microscopy and computer vision analysis. Our algorithms can reliably and automatically extract beating signal characteristics such as frequency, irregularity, and duration through image analysis of cardiomyocyte motion. Through a correlative study with MEA, we demonstrate that a non-invasive measurement of QT interval can be derived from the duration of visible cellular motion that occurs during contraction and relaxation. We also show that our system can accurately characterize the cellular response from the addition of compounds known to modulate beating frequency and irregularity. Our measurement technique is robust, automated, and requires no physical or chemical contact with the cells, making it ideal for cardiovascular drug development and cardiotoxicity testing.
Abstract 228: Cardioprotection Of Recombinant Human Mg53 Protein In A Porcine Model Of Ischemia And Reperfusion Injury
Authors: Zhu Hua , Liu Jianxun , Zheng Yongqiu , Li Lei , Tan Tao , Park Ki Ho , Hou Jincai , Liu Zhengguo , Ma Jianjie
Published: July 2021

Rationale:
Ischemic heart disease is a leading cause of death in human population and protection of myocardial infarction (MI) associated with ischemia-reperfusion (I/R) injury remains a challenge. MG53 is an essential component of the cell membrane repair machinery that protects injury to the heart.

Objective:
We investigated the therapeutic value for using the recombinant human MG53 (rhMG53) protein for treatment of MI.

Methods and Results:
Using Langendorff perfusion of isolated mouse heart, we found that I/R caused injury to cardiomyocytes and release of endogenous MG53 into the extracellular solution. The exogenous rhMG53 protein applied to the perfusion solution could concentrate at injury sites on the cardiomyocytes to facilitate cardioprotection. With rodent models of I/R-induced MI, we established the
in vivo
dosing range for rhMG53 in cardioprotection. Using a porcine model of angioplasty-induced MI, the cardioprotective effect of rhMG53 was evaluated. Intravenous administration of rhMG53, either prior to or post ischemia, reduced the infarct size and troponin I release in the porcine model when examined at 24 hrs post reperfusion. Biochemical study showed that the systemically delivered rhMG53 localized to the acute infarct zone of the porcine heart. Echocardiogram and histological analyses revealed that the protective effects for rhMG53 observed following acute MI led to long-term improvement in cardiac structure and function in the porcine model when examined at 4 weeks post operation.

Conclusions:
Cardioprotection of rhMG53 in porcine model supports the concept that rhMG53 protein could have therapeutic value for treatment of MI in human patients with ischemic heart disease.
Abstract 230: Regional Variation in Intrinsic Mechanics of Arterial Vascular Smooth Muscle Cells in Spontaneously Hypertensive Rats
Authors: Lee Jia-Jye , Lai Lo , Qiu Hongyu , Costa Kevin D
Published: July 2021

Aims:
An increased aortic stiffness is a fundamental manifestation of hypertension (HT). Our previous study showed that intrinsic mechanical properties of aortic vascular smooth muscle cells (VSMC) are an important contributor to the increased large artery stiffness in HT. However, whether VSMC mechanics of smaller arteries is also altered in HT remains unknown. The goal of this study is to test our hypothesis that the VSMC elastic and adhesive properties vary along the arterial tree reflecting the regional heterogeneities of physiological loading and geometric properties of the artery wall.

Methods and Results:
Primary VSMCs were isolated from the thoracic aorta (TA) and renal artery (RA) of adult spontaneously hypertensive rats (SHR) (16 weeks old, male) and age-matched Wistar-Kyoto normotensive (WKY) rats. Atomic force microscopy (AFM) was used to measure mechanical properties of individual VSMC at 37°C. Local apparent elastic modulus (Eap) was determined using Hertz contact analysis for a cone to model the indentation force curve, and maximum adhesion force (Fad) was obtained from the retraction force curve; results were shown as mean (±SD) (n=10 cells per condition) and compared using two way ANOVA. Eap of VSMCs from the TA was significantly higher in SHR (7.0± 1.3 kPa) vs. WKY (5.3 ± 1.5 kPa; p < 0.001) and Fad was significantly larger in SHR (39.9 7.2 pN) vs. WKY (30.6 10.9 pN; p < 0.01). No difference was found between SHR and WKY in VSMCs from renal artery in terms of Eap (4.5 0.8 kPa vs. 4.8 0.9 kPa; p = 0.59) and Fad (23.1 5.2 pN vs. 28.0 7.0 pN; p = 0.17). Although no difference was observed in cell shape represented by the cellular length:width ratio (p > 0.25), stiffness and adhesion of VSMC from TA were significantly higher vs. RA in SHR (p<0.0001) but not in WKY (P>0.35), indicating that the altered mechanics of VSMC in hypertension is more prominent in the large conduction vessel compared to the small artery.

Conclusion:
Intrinsic stiffness and adhesion of isolated VSMC are elevated preferentially in the thoracic aorta of SHR rats; the regional variations may associate mechanistically with increased aortic tissue stiffness in hypertension.
Abstract 231: Uncovering the Mechanism and Function of Newly Discovered KCNQ1-Transporter Complexes
Authors: Neverisky Daniel L , Abbott Geoffrey W
Published: July 2021

The KCNQ1 voltage-gated potassium channel is essential for human ventricular repolarization, permitting potassium efflux from excited cardiomyocytes to end each action potential and repolarize the heart. In cardiomyocytes, KCNQ1 is modulated by interaction with beta-subunits from the KCNE gene family, each of which significantly alters KCNQ1 channel function. KCNQ1 mutations are the most common identified genetic basis for Long QT syndrome (LQTS) and are also associated with lone atrial fibrillation (AF). The sodium-dependent myo-inositol transporter 1 (SMIT1) mediates cellular uptake of myo-inositol, an essential osmolyte that also represents an important substrate for phosphatidylinositol signaling pathways that regulate a plethora of ion channels including those essential for human cardiac function. We recently discovered that KCNQ1 can form heteromeric, co-regulatory complexes with Na+-coupled solute transporters including SMIT1, SMIT2 and glucose transporter SGLT1. These findings represent the first reported example of formation of an ion channel-solute transporter complex. Having discovered KCNQ1-SMIT1 complexes in mouse choroid plexus epithelium, we are currently investigating whether these types of complexes occur in the heart, how their function is altered by the various cardiac-expressed KCNE regulatory subunits or by arrhythmia-associated mutations, and which parts of KCNQ1 coordinate complex formation. Here, we present evidence of KCNQ1-SMIT1 co-assembly in pig heart based on co-immunoprecipitation experiments. Using KCNQ1-KCNQ4 chimeras we also begin to define which specific regions of KCNQ1 are required for complex formation with SMIT1. Finally, we present data showing the effects of SMIT1 on complexes formed by KCNQ1 and KCNE1, 2 and 3. KCNQ1-transporter complexes provide a potential hub for electrochemical crosstalk in normal cardiac function and in arrhythmogenesis.
Abstract 233: Engineering Growth Factors For Cardiomyocyte Survival and Regeneration Following Ischemic Injury
Authors: Stowe Timothy R , Eshghi Shawdee , Jennings-Antipov Laura D , Zhang Yan , Gip Phung , Onsum Matthew D
Published: July 2021

Background:
Despite the benefits of reperfusion therapy after myocardial infarction (MI), one-year mortality and morbidity rates remain high. Silver Creek Pharmaceuticals is engineering a new class of protein therapeutics, termed Smart Growth Factors (SGFs), designed to act directly on cardiomyocytes (CMs) to promote survival and regeneration after ischemia/reperfusion (I/R) injury. Our approach is to maximize pro-survival growth factor signaling in damaged cells by creating targeted molecules with optimized pharmacokinetics and dynamics.

Methods and Results:
Computer model simulations guided the design of a panel of SGFs comprising various growth factors, targeting arms, half-life modulators, and linkers. SGF constructs were cloned and subsequently produced using a HEK 293F expression system. This panel was screened in vitro for signaling pathway activation and target affinity. The top hits were assayed for pro-survival activity in a chemical injury model in human CMs, where a targeted SGF significantly reduced caspase activation (p<0.0084; SGF-treated 3.82±0.64-fold, IGF1-treated 16.25±3.6-fold) Based on these data and additional simulations, a class of SGFs that utilized annexin V to target IGF1 to apoptotic cells via phosphatidylserine binding was selected for screening in animal models. In vivo, these SGFs extended pAKT signaling in heart tissue compared to wild-type IGF1 and had increased accumulation in the infarcted region of mouse hearts after MI. Preliminary data suggests that a single low intravenous dose (16 pmol) of SGF delivered one hour after LAD ligation could significantly reduce scar size in animals assessed at 12 weeks post MI (p<0.05; SGF-treated 9.3±1.3%, n=10; vehicle 18.1±3.13%, n=8). Finally, SGF treatment at the time of reperfusion in a rat I/R model was able to significantly reduce infarct size (p<0.05; SGF-treated 27.0±1.8% n=7; vehicle 33.6±1.92% n=8).

Conclusions:
This work demonstrates an enhanced ability of SGFs in preventing apoptosis in vitro and reducing infarct sizes in animal models of MI. By taking a systems biology approach to SGF engineering, Silver Creek is developing cardioprotective therapies with the hope of improving patient health and addressing unmet medical need.
Abstract 24: Bone Marrow Cells Transdifferentiate into Cardiomyocytes and Repair the Infarcted Heart
Authors: Czarna Anna , Kim Junghyun , Sanada Fumihiro , Signore Sergio , Cannata Antonio , Rota Marcello , Leri Annarosa , Anversa Piero
Published: July 2021

A major controversy concerns whether c-kit-positive bone marrow cells (c-kit-BMCs) can acquire cell lineages different from their organ of origin. Technical challenges and variability in the protocols employed for cell isolation and detection of donor-derived structures may account only in part for the conflicting results obtained in different laboratories. We raised the hypothesis that c-kit-BMCs are functionally heterogeneous and possess distinct transdifferentiation potential. To address this issue, two methodologies were employed to label individual c-kit-BMCs and define their clonal fate in vivo: viral gene-tagging and fluorescent protein-based multicolor cell marking. c-kit-BMCs were infected with EGFP lentiviruses and injected in infarcted hearts. At 2 weeks, the regenerated myocardium was enzymatically digested and EGFP-labeled myocytes, endothelial cells, fibroblasts, and c-kit-cells were sorted. By employing a PCR-based method of detection of viral integrants, we searched for sites of viral insertion in the isolated cells. Common insertion sites were found in the DNA of c-kit-BMCs and specialized cells, documenting that single c-kit-BMCs transdifferentiated into multiple cell lineages. However, only specific subsets of c-kit-BMCs generated cardiomyocytes in vivo, strongly indicating that the c-kit-BMC pool is composed of myogenic and non-myogenic cells. To strengthen these observations, FACS-sorted c-kit-BMCs were simultaneously transduced with 3 lentiviruses, each encoding red, green or blue (RGB) fluorescent proteins. Different combinations of inserted vectors in individual c-kit-BMCs resulted in a variety of mixed colors, each indicative of the development of clonal populations. RGB-infected c-kit-BMCs were delivered to infarcted rats and gave rise to homogeneously colored colonies containing fluorescently labeled myocytes and vascular cells. The homogenous pattern of expression of the fluorescent proteins documented that the regenerated structures derived from the expansion and transdifferentiation of single c-kit-BMCs. Our findings demonstrate that c-kit-BMCs are functionally heterogeneous and have a different ability to survive, engraft, and transdifferentiate in the ischemic heart.
Abstract 242: Subcutaneous and Visceral Adipose-Derived Stem Cells Have Similar Biological Properties and Both Improve Cardiac Function of Infarcted Rat Hearts
Authors: Chi Chao , Xiang Bo , Deng Jixian , Wang Fei , Natarajan Kanmani , Lin Hung-Yu , Liu Hongyu , Lin Francis , Freed Darren H. , Arora Rakesh C. , Tian Ganghong
Published: July 2021

Background:
Adipose stem cells (ASC) from subcutaneous and visceral adipose tissues have been studied individually. However, it is unclear whether ASC from the two sources have different biological properties and, more importantly, whether one sub-type of ASC is more effective in treatment of CHF. This study was designed to address these concerns.

Methods:
Morphology, yield, proliferation, surface markers, and cytokine secretion of rat subcutaneous ASC (S-ASC) and visceral ASC (V-ASC) were analyzed. A rat model of myocardial infarction (MI) was established by occlusion of the LAD. 7 days after MI, S-ASC (n = 11), V-ASC (n = 11), and cell culture medium (Control, n = 7) were injected into the infarct rim, respectively. Cardiac function of the infarcted hearts was monitored with MRI for 6 months.

Results:
Both S-ASC and V-ASC exhibited a fibroblast-like morphology and expressed stromal cell markers (CD29, CD90 and CD105). No significant expression of hematopoietic markers (CD11b, CD34 and CD45) was found. Under appropriate conditions, both cells could differentiate to adipocyte- and osteocyte-like cells. Both of them expressed a significant level of HGF, IGF-1 and VEGF. As to their differences, V-ASC had approximately 3-times greater cell yield and a lower colony-formation rate (9.8±1.0% vs.13.5±2.6%) relative to S-ASC. In contrast, S-ASC showed a significantly greater growth rate (Doubling Time: 17.9 h vs. 26.0 h) relative to V-ASC. Both S-ASC and V-ASC-treated hearts showed a significantly greater left ventricular ejection fraction (LVEF, 58.3% and 56.7%) than the control group (LVEF, 47.2%) at end of 6 months. LVEF between the two ASC-treated groups was not significantly different. Finally, the implanted stem cells were readily detected in vivo with MRI for at least 6 months. Myocardial tissue sections showed existence of ASC and their locations matched with MRI signals.

Conclusions:
S-ASC and V-ASC share several biological characteristics. Both provide comparable significant improvement on cardiac function. Moreover, these implanted cells can be reliably tracked for at least 6 months using MRI. We conclude that the S-ASC and V-ASC are equally effective for treatment of heart failure.
Abstract 244: Cytoprotective Effect of Growth Hormone Releasing Hormone Agonist in Cardiac Stem Cells
Authors: Florea Victoria , Majid Sonia S , Kanashiro-Takeuchi Rosemeire M , Block Norman L , Schally Andrew V , Hare Joshua M , Rodrigues Claudia O
Published: July 2021

Background:
Our group has previously shown that growth hormone releasing hormone receptor agonists (GHRHR-A) improve cardiac performance in heart failure models and reverse remodeling. This effect was associated with an increase in the number of c-kit+ cardiac stem cells (CSCs), suggesting that this agonist might have an effect on these cells.

Methods and Results:
We investigated the expression of GHRH receptor (GHRHR) in CSCs of different species by flow cytometry analysis. GHRH-R is expressed in 96-98% of CSCs isolated from mouse, rat and porcine. Results were compared to GHRHR expression in HeLa and MCF7, and T47D cell lines, positive and negative controls, respectively. To determine if GHRHR activation can improve CSCs self-renewal, we tested the effect of agonists on porcine CSCs proliferation. The rate of cell division was increased 2-fold with JI38 (GHRHR-A) treatment (3.4 ± 0.7) vs. vehicle control (1.7 ± 0.2) (p<0.05). Pre-treatment of CSCs with the GHRHR antagonist MIA-602, showed a trend toward reversal of the JI38 agonistic effect on proliferation rate (2.2 ± 0.6). These studies were further extended to other GHRHR agonists. In addition to JI38, MR356 and MR409, both of which showed significant increase in CSCs proliferation relative to vehicle control, by 20 ± 5.7%, 37 ± 8.5% and 36 ± 12.2%, respectively (p<0.05). The protective effect of JI38 on porcine CSCs survival was determined under oxidative stress generated by hydrogen peroxide exposure. Pre-treatment of CSCs with JI38 prior to peroxide exposure significantly reduced cell death by 33 ± 2.2% (p<0.02). Similar effects were observed for MR356, which decreased cell death by 12 ± 8.6% (p<0.03). Furthermore, we found that the effect of GHRHR-A on CSCs proliferation was completely reversed by inhibitors of the ERK, PI3K and Akt pathways (p<0.05).

Conclusion:
These findings confirm for the first time the expression of GHRHR in CSCs. GHRHR-A promotes CSCs proliferation and enhance survival. GHRHR-A effects on CSCs proliferation are mediated through activation of ERK, PI3K and AKT pathways. Accordingly, activation of GHRHR signaling pathways represents a novel therapeutic approach to protect and stimulate endogenous CSC population, promoting cardiac repair.
Abstract 247: Heparanase-overexpressed Mesenchymal Stem Cells Enhances Angiogenesis Via Hypoxia-inducible Factor-2α/Vascular Endothelial Growth Factor Receptor-2 Pathway
Authors: Hu Xin Yang , Zhang Ling , Jin Jing , Cheng Huiqiang , Chen Han , Wang Jian-an
Published: July 2021

Background:
Heparanase is an endoglycosidase involved in the cleavage of the heparin sulfate and plays important role in tumor metastasis and angiogenesis. Here we showed heparanase - overexpressed mesenchymal stem cells (MSCs) promoted angiogenesis in rat model after femoral artery ligation, and its underlying mechanism was via endothelial cells migration by Hypoxia-Inducible Factor-2α (HIF-2α) / vascular endothelial growth factor receptor-2 (Flk-1) pathway and collateral formation.

Methods:
MSCs were transduced with heparanase (MSC
heparanase
), control cells were either nontransduced (MSC
WT
) or transduced with empty vector (MSC
Null
). The effect of human umbilical vein endothelial cells (HUVECs) migration, tube formation and the role for heparanase were determined by transwell, matrigel and arterial rings asaay after co-culture with MSC
heparanase
, MSC
Null
and MSC
WT
conditioned medium. Moreover, right femoral artery was occluded in rats receiving PBS or MSC
Null
or MSC
heparanase
, blood flow recovery was assessed by laser-Doppler imaging (LDI) before surgery, immediately after operation, as well as 3, 7 and 14 days after occlusion, capillary density was measured at 14 days after occlusion.

Results:
In vivo
, the group receiving MSC
heparanase
had a fast recovery of blood flow at 7 days after occlusion and more capillary densities at 14 days after occlusion.
In vitro,
HUVECs treated with MSC
heparanase
conditioned medium exhibited increased formation of capillary-like structures and promoted migration, compared with HUVECs treated with MSC
Null
conditioned medium. All effects of MSC
heparanase
conditioned medium were abolished by knockdown of HIF-2α and flk-1 with a selective shRNA and were absent in HUVECs. Mechanism studies revealed that heparanase increased the cellular accumulation of HIF-2α protein and affects its nuclear translocation, which regulates flk-1 and its downstream proteins: p38 and HSP27 expression.

Conclusions:
These findings identify heparanse as a key regulator in MSCs induced angiogenesis and collateral formation. Heparanse up-regulates HIF-2α and mediates Flk-1/p38/HSP27 pathway, which is necessary for angiogenesis and collateral artery network.
Abstract 252: Generation of a Mouse ESC Reporter Line to Identify Purkinje-like Conduction System Cells
Authors: Maass Karen , Lu Jia , See Fiona , Kang Guoxin , Delgado Camila , Fishman Glenn I
Published: July 2021

We have previously demonstrated that contactin2 (Cntn2) protein is enriched in Purkinje cells of the adult cardiac conduction system (CCS). Here we present a mouse embryonic stem cell (mESC) reporter line to identify Purkinje-like cardiomyocytes in vitro.

Methods and Results:
Dual reporter Purkinje cell mESC were generated from Cntn2-eGFP blastocysts, transduced with αMHC-mCherry lentivirus. The mESC expressed markers of pluripotency (Oct3/4; Sox2, Nanog) and differentiated into all three germ layers (Nestin, a-fetoprotein, Brachyury). Cultures differentiated under serum-free, cardiogenic conditions were enriched in cardiomyocytes (n=5), consisting of single positive aMHC-mCherry [57.76±3.7%] and double positive αMHC-mCherry/Cntn2-eGFP cells [1.9±0.9%]. Compared with eGFP negative cardiomyocytes (n=15), double positive cells (n=11) revealed electrophysiological characteristics consistent with Purkinje-like derivatives, including increased peak sodium currents (180.3±20.5 pA/pF vs. =-118.0±21.2pA/pF), faster upstroke velocities (133.7±12.3 V/s vs.60.0±13.2V/s) and elongated action potential duration (APD90=170.2±17.5ms vs. APD90=120.6±17.3ms). Calcium imaging demonstrated chronotrop regulation and spontaneous Ca2+ oscillations in mCherry+eGFP+ cells. Immunofluorecence analysis demonstrated endogenous expression of Purkinje cell markers, including Cntn2, Cx40 and Troponin-T, in double positive cells. Quantitative real-time PCR analysis of double positive cells verified cardiomyocyte-specific transcript expression [Mlc2v: 3.8x106-fold (p<0.05); Nkx2.5: 178-fold (p<0.05) compared to double negative cells]. Moreover, double positive cells expressed significantly elevated levels of CCS-specific transcripts compared to mCherry-single positive cells [Cntn2: 31-fold (p<0.001); Cx40: 878 -fold (p<0.01); Cav3.1: 11-fold (p<0.05); HCN4: 7-fold (p<0.001)].

Conclusion:
The dual reporter Purkinje cell mESC reporter line permits identification and enrichment of ventricular CCS derivatives. [Supported by NYSTEM contracts #C024327 and #C028115 to GIF].
Abstract 253: The Presence of Coronary Artery Disease Influences Number and Function of Stem Cells in the Bone Marrow
Authors: Nollet Evelien , De Bock Dina , Rodrigus Inez R , Hoymans Vicky Y , Vrints Christiaan J , Van Craenenbroeck Emeline M
Published: July 2021

Purpose:
Despite the observed therapeutic benefits of autologous bone marrow (BM)-derived stem cell transplantation in patients with ischemic heart disease, the efficacy of this approach could be hampered by BM dysfunction. We investigated whether BM cellularity and function is affected by coronary artery disease (CAD).

Methods & Results:
BM samples were obtained peri-operatively from 26 CAD patients, undergoing coronary artery bypass surgery (LVEF 54±16%), and 6 controls, undergoing mitral valve surgery (LVEF 50±12%; age 59±10yrs). CAD patients were stratified according to their Syntax score (mild ≤15, age 61±10yrs; and moderate CAD >15, age 63±8yrs; stratification based on median score), which is used to assess complexity of coronary lesions. In vitro functional analysis of isolated BM-derived mononuclear cells (BM-MNC) revealed a significant impairment of migratory capacity towards SDF-1α and VEGF in patients with moderate CAD (25.71±7.3%) compared to controls (33.82±8.3%; p=0.042) and patients with mild CAD (34.76±7.8%; p=0.007). Hematopoetic stem cells (HSC, CD45dimCD34+SSClow) were reduced in patients with moderate CAD (8178±5530 HSC/106 BM-MNC; p=0.014) and mild CAD (10655±5489 HSC/106 BM-MNC; p=0.054) compared to controls (16220±6126 HSC/106 BM-MNC). An inverse correlation was found between age and the number of granulocyte-macrophage colony forming units (r= −0.408; p=0.048), burst forming units erythroid (r= −0.458; p=0.028) and HSC (r=-0.356; p=0.046). Furthermore, our data revealed a relation between reduced renal function (CKD-EPI eGFR, 81.2±19 ml/min) and reduced number of HSC (r=0.480; p=0.011) and endothelial progenitor cells (EPC, CD45dimCD34+KDR+; r=0.522; p=0.008).

Conclusions:
Migratory capacity of BM-MNC and the number of HSC are reduced in patients with CAD, which is more pronounced in more complex CAD. In addition, age and renal function emerge as relevant determinants on BM function and stem cell populations. Therefore, these factors should be taken into account when assessing benefits of autologous stem cell therapy.