bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 142: Role of CK2α in Heart Tube Proliferation
Authors: Dominguez Isabel , Roman Degano Irene , Macias Alvarez Laura , Revuelta-Cervantes Jesus
Published: July 2021

Congenital heart disease (CHD) groups a number of structural malformations of the heart and associated blood vessels present at birth. CHD is the most common birth defect, arise from defective cardiogenesis, and can be due to genetic and/or environmental factors that affect cellular processes such as proliferation during heart formation. The highly conserved serine-threonine kinase, CK2α, has been implicated in proliferative processes, including cancer, and it is a target of environmental toxins. To test whether CK2α plays a role in cardiogenesis, we used a CK2α deficient mouse. CK2α-/- mice die around embryonic day (E)11 and showed dysmorphic outflow tracts (OFT), hypoplastic right ventricles (RV) and poorly developed ventricular trabeculations. These defects, the probable cause of the lethality, could be due to deficient proliferation. Thus, we tested whether reduced CK2α expression leads to proliferative signal defects during cardiogenesis.

We used three or more pairs of somite-matched wild-type and CK2α-/- embryos for the analysis of proteins by immunofluorescence and immunoblot, and of transcript expression by RT-qPCR and in situ hybridization. At E9.5 and E10.5, the number of Isl1+ cells in the OFT and the levels of Isl1 transcript in the heart of CK2α-/- embryos was no different from wild-type. These data suggest that the mechanism underlying the OFT and RV defects is different from progenitor incorporation defects. CK2α deficient hearts expressed pan-cardiac gene and protein markers normally at E9.5 and E10.5; and apoptosis was not affected, indicating that the heart malformations may be due to proliferative defects. Indeed, mitotic rates were decreased throughout the heart in CK2α mutants compared to wild-type embryos at E9.5 and E10.5; but a mitotic defect was not apparent at E8.5. The observed decrease in proliferation correlated with decreased expression of cardiac proliferative markers such as n-myc and decrease in proliferative signal pathways in the heart tube.

In conclusion, these data show that, from E9.5 on, CK2α is necessary for proliferation and proliferative signal pathway activation in the heart tube; and that cellular mechanisms other than progenitor incorporation may play a key role in OFT and RV defects.
Abstract 15: Remote Ischemic Preconditioning Confers Late Protection Against Myocardial Ischemia-Reperfusion Injury in Mice by Upregulating Interleukin-10
Authors: Cai Zheqing , Parajuli Nirmal , Zheng Xiaoxu , Becker Lewis
Published: July 2021

Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia-reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of IL-10. Mice were exposed to lower limb RIPC or sham leg ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120), accompanied by increased phosphorylation of Akt and eNOS in the heart. These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies. Moreover, in IL-10 KO mice, RIPC was no longer able to provide cardioprotection against IR injury. IL-10 receptors were expressed in cardiomyocytes under basal conditions. Stimulation with exogenous IL-10 increased Akt phosphorylation and decreased myocardial infarct size after I-30/R-120 in isolated perfused hearts. Wildtype mice with RIPC demonstrated increased plasma IL-10 levels, while in the preconditioned gastrocnemius muscle, the phosphatase PTEN was inactivated and expression of IL-10 was increased through Stat3. Myocyte-specific PTEN inactivation led to increased Stat3 phosphorylation and IL-10 protein expression in the gastrocnemius muscle. Taken together, these results suggest that RIPC induces late protection against myocardial IR injury by increasing expression of IL-10 in the remote muscle, followed by release of IL-10 into the circulation, and activation of protective signaling pathways in the heart. This study provides a scientific basis for the use of RIPC to confer systemic protection against IR injury.
Abstract 151: A Polymorphic T-Rich Element in ATP1B1 Is Associated with Blood Pressure and Regulates Alternative Polyadenylation
Authors: Prasad Megana , Bhalla Kavita , Pan Zhenhua , O'Connell Jeffrey R , Ji Zhe , Weder Alan , Chakravarti Aravinda , Tian Bin , Chang Yen-Pei C
Published: July 2021

Genome-wide linkage and association studies of hypertension (HTN) aim to uncover loci that are involved in blood pressure (BP) regulation and the pathophysiology of HTN. However, even when such loci are successfully identified, the mechanism by which the implicated genes increase HTN susceptibility is not well understood. ATP1B1, which encodes the beta subunit of Na+, K+ ATPase, a co-transporter involved in multiple BP-regulating physiological processes, is located within a well-established BP linkage peak. Although a single nucleotide polymorphism (rs12079745) in the 3’UTR of ATP1B1 has previously been shown to be associated with BP levels, the molecular mechanism underlying this association is unknown. We identified a multi-allelic T-rich element (TRE) in the 3’UTR of ATP1B1 that varies in length and sequence composition (T22-27 and T12GT3GT6). The 3’UTR of ATP1B1 contains 2 functional polyadenylation signals and the TRE is downstream of the proximal site (A2). Because U-rich elements (UREs) in 3’UTRs are known to play a role in the cleavage and polyadenylation of mRNA, and ATP1B1 transcripts with shorter 3’UTRs are translationally more efficient, we hypothesized that alleles of this TRE might influence ATP1B1 expression by regulating alternative polyadenylation. Consistent with our hypothesis, the A2-polyadenylated mRNA isoform was more abundant in human kidneys with at least one copy of T12GT3GT6 than in those homozygous for the T22-27 alleles. Functionally, in vitro, we demonstrated that the T12GT3GT6 allele has greater luciferase activity than the T22 allele and that the T12GT3GT6 allele shows increased polyadenylation at the A2 site than the T22 allele. In addition, the TRE element is associated with systolic BP in European Americans of the GenNet network of the NHLBI Family Blood Pressure Program, with the T12GT3GT6 allele being associated with higher BP (effect size = 2.4 mmHg SBP, P = 0.003). Sharing strong linkage disequilibrium with rs12079745, this TRE is likely the functional site underlying the original association. In summary, we have identified a novel multi-allelic TRE in the 3’UTR of ATP1B1. Alleles at this site are associated with HTN and may mediate their effect on BP by regulating polyadenylation of the ATP1B1 mRNA.
Abstract 155: TGF-β1 Positively Modulates Cell-Metal Interaction in Cardiovascular Applications
Authors: Alavi S Hamed , Kheradvar Arash
Published: July 2021

Introduction:
The interaction between a metallic implant and local cells in the cardiovascular system determines the fate of the implant. We have recently developed a hybrid scaffold for tissue engineering of valves, which consists of an extra thin layer of Stainless Steel mesh tightly enclosed by three distinct layers of cells. We have examined the effect of TGF-β1 on degree of cell attachment to the metal mesh.

Methods and Results:
By using a 3D cell culture method a Stainless Steel mesh with desired size and thickness was enclosed through cultured layers of cells similar to a heart valve leaflet pattern. Human aortic smooth muscle cells and adventitial fibroblast/myofibroblast cells were used to fulfill the role of valvular interstitial cells, and human umbilical vascular endothelial cells were used to cover the construct in place of the valvular endothelial cells. The layers were seeded in sequence on a three dimensional, porous biological scaffold comprised of bovine type I collagen gel mixture. The effect of TGF-β1 supplementation at proper time-steps on the cultured hybrid tissue after two months of cell culture was determined. TGF-β supplementation resulted in significant increase in proliferation, and extra ingrowths into the outer portions of the tissue. DAPI staining of nuclei showed that the number of cells at the surface of the mesh progressively increased due to TGF-β, and eventually formed a thicker tissue around the mesh

Conclusions:
We have demonstrated the effects of TGF-β1 on cell attachment and tissue development by using a hybrid tissue material that helped us study the direct cell-metal interactions
in-vitro.
Abstract 16: Genomewide Association Study of Isoproterenol-Induced Heart Failure in a Large Mouse Panel
Authors: Rau Christoph D , Wang Jessica J , Ren Shuxun , Wang Yibin , Lusis Aldons J
Published: July 2021

Heart failure is highly heterogeneous and association studies in humans have yielded few insights into its genetic basis. We have developed a resource, the Hybrid Mouse Diversity Panel, to efficiently perform association studies on complex diseases in a mouse model. Each strain within the panel has been densely genotyped and the panel displays significant baseline inter-strain variation. We used the panel to explore isoproterenol (a β-adrenergic agonist) induced heart failure.

Eight week old females from 105 unique inbred strains (average N = 6.7) were divided into control (average N = 2.5) and treated (average N = 4.1) cohorts. Treated mice received 20 μg/g/day of drug through an abdominally implanted Alzet micropump. All mice underwent echocardiography to assess left ventricular function both before and at weekly timepoints after treatment. At three weeks, all mice were sacrificed. Hearts (sectioned by chamber) lungs, liver and adrenal glands were weighed before storage for further analysis. Phenotypes were analyzed using the Efficient Mixed-Model Association (EMMA) algorithm to correct for population substructure. Promising gene candidates were tested on zebrafish morpholino models to assess any phenotypic effects.

Here we report initial findings from the panel. Chronic β-adrenergic stimulation results in marked variations in both weight and echocardiographic measurements. EMMA analysis of the data revealed several dozen putative peaks, including several repeated peaks in correlated phenotypes as well as replication of previously reported loci in both human and mouse studies. Further analysis of these peaks will shed light on the genetics underlying heart failure.
Abstract 161: ABCB10 Plays a Role in Heme Synthesis and Mitochondrial δ-Aminolevulinic Acid Export
Authors: Khechaduri Arineh , Bayeva Marina , Ardehali Hossein , Wu Rongxue
Published: July 2021

Introduction:
Heme is essential for cardiovascular health, as it is a component of myoglobin, cytochrome P450, electron transport chain complexes, and antioxidant enzymes. The eight steps of heme synthesis are well characterized; however, it is not known how the synthetic intermediates are transported between mitochondria and cytosol. We hypothesized that the mitochondrial ATP-binding cassette protein-B10 (ABCB10) plays a role in heme synthesis by mediating the export of δ-aminolevulinic acid (ALA) out of the mitochondria.

Results:
Lentiviral shRNA-based knockdown of ABCB10 in H9c2 cardiac myoblasts significantly reduced cytosolic and mitochondrial heme levels and the activities of heme-containing proteins (catalase, peroxidase and complex IV), while their mRNA levels remained unchanged. The observed heme deficiency was not due to a defect in heme synthesis, iron incorporation, or enhanced heme degradation, as ABCB10 shRNA had no effect on the expression of enzymes mediating these steps. We then tested whether ABCB10 regulates mitochondrial export of ALA. Addition of exogenous ALA completely reversed the reduction in heme levels and normalized the activities of heme containing enzymes in cells with ABCB10 knockdown. However, overexpression of ALA synthase 1 (ALAS1), an enzyme catalyzing the production of ALA in the mitochondria, resulted in increased heme levels at baseline, but failed to restore heme content in ABCB10 shRNA-treated cells. To examine the role of ABCB10 in cardiac disease, we measured ABCB10 expression in the hearts of mice subjected to myocardial infarction and in explanted human hearts with ischemic cardiomyopathy. ABCB10 protein was significantly elevated in mouse and human hearts, and adenoviral overexpression of ABCB10 in H9c2 cardiac myoblasts significantly attenuated the production of reactive oxygen species (ROS), suggesting the protective role for ABCB10 in heart disease.

Conclusions:
Our studies place ABCB10 between the mitochondrial synthesis of ALA by ALAS1 and the cytosolic conversion of ALA into heme, suggesting that ABCB10 plays a role ALA export out of the mitochondria. Furthermore, its overexpression exerts protective effects against oxidant induced cell death in cardiac cells.
Abstract 162: Cardiac Overexpression of Senescence Marker Protein 30 Inhibits Angiotensin II-Induced Cardiac Hypertrophy and Remodeling
Authors: Misaka Tomofumi , Suzuki Satoshi , Miyata Makiko , Kobayashi Atsushi , Saitoh Shu-ichi , Takeishi Yasuchika
Published: July 2021

Backgrounds:
Senescence marker protein 30 (SMP30) was originally identified as an important aging marker protein, and assumed to behave as an anti-aging factor. Previously, we demonstrated that deficiency of SMP30 exacerbates angiotensin II (Ang II)-induced reactive oxygen species (ROS) and cardiac adverse remodeling, suggesting that SMP30 may have a protective role in the heart. Thus, this study aimed to test the hypothesis that up-regulation of SMP30 inhibits cardiac hypertrophy and remodeling in response to Ang II.

Methods:
We generated transgenic (SMP30-TG) mice with cardiac-specific overexpression of SMP30 gene using α-myosin heavy chain promoter. SMP30-TG mice and wild type littermate (WT) were subjected to continuous Ang II infusion (800 ng/kg/min).

Results:
After 2 weeks, heart weight was significantly lower in SMP30-TG mice than in WT mice (P<0.01). Echocardiography revealed that calculated left ventricular mass and E/e’ were lower in SMP30-TG mice than in WT mice (P<0.01 and P<0.05, respectively), suggesting that diastolic function was preserved in SMP30-TG mice. Histological analysis showed that the degree of cardiac fibrosis was significantly decreased in SMP30-TG mice than in WT mice (P<0.05). Dihydroethidium staining demonstrated that generation of ROS was reduced in SMP30-TG mice compared with WT mice (P<0.05). Furthermore, the numbers of senescence-associated β-galactosidase-positive cardiomyocytes were decreased in SMP30-TG mice compared to WT mice (P<0.05). In addition, p21 mRNA level was significantly suppressed in SMP30-TG mice compared to WT mice (P<0.01).

Conclusions:
This study demonstrated cardiac-specific overexpression of SMP30 inhibits Ang II-induced cardiac hypertrophy and remodeling. These findings suggested that SMP30 has a cardio-protective role with anti-oxidative and anti-aging effects. Up-regulation of SMP30 might be a new strategy to approach senescent cardiac diseases and attenuate the development of heart failure particularly with hypertension.
Abstract 163: Interleukin-13 Expression Attenuates Left Ventricular Dilation and Mortality After Myocardial Infarction
Authors: Hofmann Ulrich , Knorr Susanne , Weirather Johannes , Frey Anna , Frantz Stefan
Published: July 2021

Background:
Monocyte infiltration and extracellular matrix breakdown/ de novo formation critically influence wound healing and remodeling after myocardial infarction (MI). Interleukin-13 (IL-13) has potent pro-fibrotic properties and is a differentiation factor for monocytes/ macrophages. Therefore, we studied whether IL-13 affects myocardial wound healing and remodelling after MI.

Methods and results:
MI was induced by permanent ligation of the left coronary artery in WT and IL-13-/- mice. Expression of IL-13 was upregulated in the myocardium after MI. IL-13 expression was in part dependent on angiotensin II signalling as in vivo treatment with the Angiotensin-receptor blocker losartan attenuated myocardial IL-13 expression in response to MI. Intracellular FACS analysis of myocardial leukocytes further demonstrated IL-13 expression in a subset of myeloid CD11b+ cells within the myocardium. IL-13-/- mice showed higher mortality than WT mice after MI without difference in infarct size. Echocardiography further demonstrated increased left ventricular dilation in IL-13-/- when compared to WT mice. Histological analysis of the scar collagen did not reveal a difference between WT and IL-13-/- mice. FACS analysis of leukocytes from the infarct zone showed less MHC-II+ CD11b+ cells within the myocardium of IL-13-/- mice on day 7 indicating that IL-13 is required for the differentiation of heart infiltrating monocytes.

Conclusions:
Expression of IL-13 in response to MI beneficially modulates wound healing after MI.
Abstract 164: MicroRNA-29c Reduces Serca2 Expression During Myocardial Hypoxia
Authors: MacCannell Keith A , Shohet Ralph V , Walton Chad B
Published: July 2021

The cardiac response to a hypoxic challenge is coordinated by the transcription factor Hypoxia Inducible Factor-1 (HIF-1), which drives the transcription of a large number of genes pertinent to angiogenesis, metabolism, and calcium cycling. Expression of HIF-1alpha that has been mutated to remove oxygen regulation recapitulates many of the changes in gene expression seen in hypoxia. It also results in reversible cardiac dysfunction associated with marked decrease of Sarco-Endoplasmic Reticulum Calcium-Dependent ATP-ase (SERCA2). This decrease in SERCA was more rapid and more substantial than a moderate decrease seen in the mRNA for this protein.

In this study, we tested the hypothesis that the decrease in SERCA2 is mediated by the up-regulation of microRNA (miRNA) in the heart. We identified 30 miRNAs that are dysregulated upon induction of oxygen stable HIF1 expression. Three of these have evident binding sites in the 3’ untranslated region of the SERCA2 gene. Transient co-transfection of a mimic molecule for these three miRs with a luciferase reporter construct that includes the 3’ UTR of SERCA2 showed that mi-29c produced a substantial decrease in reporter expression. This effect is abolished by the mutagenesis of the predicted target site within the 3’UTR. Similarly, transfection of this mimic molecule into a cell line that expresses SERCA2 results in a reduction in SERCA2 protein levels. An antagomir to this miR rescues the reduction in SERCA seen with HIF expression. We conclude that miR-29c, is responsible for an important portion of the down-regulation of SERCA2, and thus the contractile dysfunction, seen in hypoxia.
Abstract 171: Plasma Hypoosmolarity After Birth Promotes Closure of the Ductus Arteriosus
Authors: Aoki Rika , Yokoyama Utako , Ichikawa Yasuhiro , Kumagaya Shun , Iwasaki Shiho , Nishimaki Shigeru , Seki Kazuo , Yokota Shumpei , Minamisawa Susumu , Ishikawa Yoshihiro
Published: July 2021

Background:
Changes in environmental conditions after birth, such as increased oxygen tension, are known to regulate the closure of the ductus arteriosus (DA). We previously found that plasma osmolarity was significantly decreased early after birth in rats, and that Transient Receptor Potential Melastatin (TRPM) 3, a hypoosmolarity sensor, was highly expressed in the rat DA compared to the aorta. We assessed the hypothesis that plasma hypoosmolarity regulated tension of the DA via TRPM3.

Methods:
Fura-2 assay was used to evaluate the change in intracellular calcium [Ca
2+
]
i
in vascular smooth muscle cells (SMCs). Tension of vascular rings was measured by a wire myograph system. Rapid whole-body freezing method was used to examine vasoconstriction in rat
in vivo
. Plasma osmolarity of human preterm infants was analyzed by freezing point depression osmometry.

Results:
We found that [Ca
2+
]
i
was increased by hypoosmolarity (270mOsm/kg) in SMCs isolated from the rat DA (DASMCs) compared to aortic SMCs (31.3% vs. 8.5% of basal 340/390 ratio
P
<0.01, n=6). When DASMCs were treated with siTRPM3, hypoosmolarity-induced [Ca
2+
]
i
elevation was attenuated by 48.7% (
P
<0.01, n=6). Hypoosmolarity (270 and 250mOsm/kg) increased the tension of rat DA (17.4% and 29.8% of 120mM of KCl, respectively,
P
<0.01, n=8). Moreover, a TRPM3 activator pregnenolone sulfate (200μ M) induced contraction of the rat DA (39.4% of KCl
P
<0.001, n=6). Conversely, when plasma osmolarity was kept higher to 322mOsm/kg by intra-peritoneal injection of 5.4% hypersaline, the DA closure was partially inhibited by 13% compared to 0.9% saline injection
in vivo
(
P
<0.05, n=11). Plasma osmolarity was decreased 2 h after birth in late preterm infants (28-35weeks’ gestation) (3.2±0.8 % reduction of cord blood,
P
<0.001, n=35), but not in early preterm infants (24-27weeks’ gestation) (1.1±1.3 % reduction of cord blood,
NS
, n=17). Furthermore, plasma osmolarity of patent DA (PDA) patients recovered to the adult level by the 2nd day of life while it still remained low in non PDA patients.

Conclusions:
These results suggest that plasma hypoosmolarity after birth promotes DA closure via TRPM3 and that keeping the plasma osmolarity within proper levels during early neonatal period would be important for DA closure.
Abstract 178: Volume Overload Induced by Mitral Regurgitation Alters Spatial Regulation of Nitric Oxide/Reactive Nitrogen Species Signaling
Authors: Liu Yuchuan , Sabri Abdelkarim , Dell'Italia Louis , Rizzo Victor , Tsai Emily J
Published: July 2021

Volume-overload (VO), as seen in regurgitant valvular disease, large myocardial infarction, and severe cardiac systolic dysfunction, triggers eccentric hypertrophy. Given that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) modulate cardiomyocyte hypertrophy, apoptosis, and cardioprotection, differential regulation of these signals may distinguish eccentric from the more commonly studied concentric hypertrophy. We recently showed that pressure-overload (PO) induces relocalization and oxidation of the NO receptor soluble guanylyl cyclase (sGC), thereby diminishing cyclase activity and cGMP cardioprotection. The effects of volume-overload on NO and cGMP signaling are unknown. We tested the hypothesis that VO induces relocalization but not oxidation of sGC, thereby disrupting spatial regulation of NO-cGMP signaling without depressing cyclase activity.

Volume-overload was established by chordal rupture-induced mitral regurgitation in dogs. We compared intracellular localization and activity of sGC in VO and control LVs (N=5 per group). Both sGC subunits were detected within and outside of caveolae-enriched lipid raft microdomains (Cav3+LR). In VO hearts, sGCβ1 fell in expression by nearly 50% and relocalized away from Cav3+LR to non-lipid raft microdomains (NLR). Despite VO-induced sGCβ1 changes, overall NO-stimulated sGC activity was preserved. An enhanced response to heme/NO-independent sGC activator BAY 60-2770 suggested that a subset of sGC was heme-oxidized in VO hearts, though to a much lesser degree than in PO hearts. As in PO hearts, Cav3+LR appear to protect sGC from heme-oxidation in VO hearts. Initial study of downstream reactive nitrogen species (RNS) and cGMP signaling supported our theory that VO altered spatial regulation of NO-cGMP signaling. Also, a trend towards increased overall tyrosine-nitration, predominantly within NLR, was observed in VO hearts.

Volume-overload shifted cardiac NO-cGMP signaling from Cav3+LR to NLR microdomains without depressing NO/heme-dependent sGC activation. These findings suggest that differential spatial regulation of NO/RNS signaling, rather than simply increased RNS signaling, might drive the distinct molecular pathophysiology of eccentric hypertrophy.
Abstract 179: Mechanical Assessment of the Drosophila Heart Reveals Conservation of Human Heart Failure Mechanisms
Authors: Kaushik Gaurav , Nishimura Mayuko , Bodmer Rolf , Cammarato Anthony , Engler Adam J
Published: July 2021

Rationale:
Current models for investigating the mechanisms of heart failure are limited by low throughput or genetic intractability. Therefore, we have developed a nanoindentation method to measure the intrinsic stiffness of the intact, living Drosophila heart tube to assess mechanical function in this rapidly aging, genetically malleable system which has been shown to experience age-related decline in cardiac function.

Objective:
Fruit fly strains universally experience decline in fractional shortening with age due to decreased diastolic diameter, though severity varies between strains. We compared a strain with significant decline in diameter with age and one without to examine the hypothesis that diastolic decline is the consequence of increase passive tension due to myocardial stiffening with age. Quantitative PCR was employed targeted towards mechanosensitive molecules known to be overexpressed in failing human hearts.

Methods and Results:
Female yw flies experience ∼18% reduction in diastolic diameter with age (59.3 at 1 week to 48.4 µm at 5 weeks of age, p < 10-3) as calculated from m-mode kymographs of beating heart tube. Nanoindentation reveals 111% increase in stiffness (1.82 to 3.85 kPa, p < 10-10) proximal to the cardiac cell-cell junctions. In contrast, w1118 flies experience ∼6% decline in diastolic diameter (74.1 to 69.5 µm, p = 0.049) and do not stiffen with age (p = 0.1923). qPCR reveals increased expression of cell-cell adhesion molecules in yw, such as vinculin and cadherin. Expression levels remain steady or decline in w1118. Further analysis reveals that w1118 experiences separation between juxtaposed muscle layers (410 nm at 1wk, 448 nm at 5wks) indicating matrix adhesion defects not observed in yw (0 nm, p < 10-7 compared to 1wk w1118; 0 nm, p < 10-8 compared to 5wk w1118).

Conclusion:
These data indicate structural changes in the fly are a consequence of adherens-junction-mediated mechanical remodeling similar to that which is observed in the failing human heart. Matrix defects and the lack of remodeling in w1118 suggest a link between costamere and adherens signaling during aging. Cardiac matrix content and adhesion signatures likely direct cardiac remodeling and function.
Abstract 18: Synoviolin, an E3 Ubiquitin Ligase, Modulates Cardiac Myocyte Size and Restores Heart Function in Hypertrophic Cardiomyopathy
Authors: Doroudgar Shirin , Völkers Mirko , Thuerauf Donna J , Bumbar Ashley , Khan Mohsin , Mohsin Sadia , Wang Wei , Respress Jonathan L , Gude Natalie , Müller Oliver J , Wehrens Xander H , Sussman Mark A , Glembotski Christopher C
Published: July 2021

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P<0.001), and decreased mTOR activity. Since calcium is a major regulator of cardiac myocyte size, we examined the effects of synoviolin gain- or loss-of-function, using AAV9-synoviolin, or an miRNA designed to knock down synoviolin, respectively. While synoviolin gain-of-function did not affect calcium handling in isolated adult myocytes, synoviolin loss-of-function increased calcium transient amplitude (P<0.01), prolonged spark duration (P<0.001), and increased spark width (P<0.001). Spark frequency and amplitude were unaltered upon synoviolin gain- or loss-of-function. Whereas SR calcium load was unaltered by synoviolin loss-of-function, SERCA-mediated calcium removal was reduced (P<0.05). In conclusion, our studies suggest that in the heart, synoviolin is 1) a critical component of proteostasis, 2) a novel determinant of cardiac myocyte size, and 3) necessary for proper calcium handling.
Abstract 181: β-Adrenergic Preconditioning Is Adenosine Mediated
Authors: Lochner Amanda , Salie Ruduwaan , Moolman Johan A
Published: July 2021

This study investigated the roles of adenosine, oxygen free radicals (ROS), the K
+
ATP
channels and the PI
3
K/PKB/Akt and ERK signal transduction pathways during the triggering and mediation phases of beta-adrenergic preconditioning (BPC)

Methods
: Using the isolated working rat heart, BPC was elicited by administration of denopamine (B
1
receptor agonist, 10
-7
M), isoproterenol (B
1
/B
2
receptor agonist, 10
-7
M) or formoterol (B
2
receptor agonist, 10
-9
M) for 5 min followed by 5 min washout. Index ischemia was 35 min regional ischemia followed by 2h reperfusion and infarct size was determined with the tetrazolium method. The role of adenosine was studied using selective adenosine receptor antagonists. Involvement of ROS, PKC, the mitochondrial K
+
ATP
channels, endogenous opioids and bradykinin was studied by using N-acetyl cysteine (NAC), bisindolylmaleimide, 5-HD, naloxone or HOE140, respectively. Wortmannin and PD98,059 were used to inhibit PI
3
K and ERK respectively. Phosphorylation of these kinases was determined by Western blot.

Results:
Preconditioning with all three B-adrenergic receptor agonists caused a significant (40-60%) reduction in infarct size and improvement in postischemic function. BPC with isoproterenol and denopamine was abolished by the adenosine A
3
receptor antagonist MRS1191 during both the triggering and mediation phases, while being effective during reperfusion only in formoterol preconditioned heart
s.
B
1
/B
2
PC was attenuated by MRS1754 (adenosine A
2B
receptor antagonist) during the triggering and mediation phases. The mediation phase of B
1
/B
2
PC was also abolished by
ZM241385
(A
2A
receptor blocker). The adenosine A
1
receptor blocker, DPCPX, was without effect. The ROS scavenger NAC caused a significant attenuation of cardioprotection induced by all three B-adrenergic receptor agonists. 5-HD, naloxone and Hoe140 were without effect on BPC. Activation of ERK and PI3K/PKB/Akt during both triggering and reperfusion phases are required for cardioprotection.

Conclusion:
BPC in rat hearts is dependent on activation of the A
3
adenosine receptors by endogenous adenosine and production of free radicals during the triggering and mediation phases while the A
2A
and A
2B
receptors participate mainly during reperfusion.
Abstract 19: Myocyte Turnover in the Aging Human Heart
Authors: OgÓrek Barbara , Hosoda Toru , Rondon Carlos , Gurusamy Narasimman , Gatti Alessandro , Bardelli Silvana , Quaini Federico , Bussani Rossana , Silvestri Furio , Daniela Cesselli , Beltrami Antonio P , del Monte Federica , Rota Marcello , Urbanek Konrad , Buchholz Bruce A , Leri Annarosa , Beltrami Carlo A , Anversa Piero , Kajstura Jan
Published: July 2021

The controversy on the growth reserve of the adult human heart has not been resolved and the extent of myocyte renewal reported by different groups varies significantly. Additionally, myocyte regeneration has been claimed to decrease with aging, although cell death is markedly enhanced in the old myocardium. Thus, the effects of age and gender on the magnitude of myocyte turnover were determined. Myocyte replication, senescence and apoptosis were measured in normal female and male human hearts collected from patients 19 to 104 years of age who died from causes other than cardiovascular diseases. Myocardial aging was characterized by a time-dependent increase in the generation of amplifying cardiomyocytes in women and men. Levels of Ki67 and phospho-H3 were comparable in the young female and male heart but differed later in life. As a function of age, the pool of amplifying myocytes was 2-fold higher in women than men, pointing to enhanced myocyte renewal in the female heart. The frequency of p16
INK4a
-positive myocytes was higher in men than in women. From 19 to 104 years of age, the time-dependent increase in senescent myocytes was 0.68% per year in women and 0.89% per year in men; the 31% higher rate of accumulation of old myocytes in the aging male heart was significant. Myocyte apoptosis occurred only in p16
INK4a
-positive cells and was consistently higher in men than in women at all age intervals. However, the increase in myocyte apoptosis with age did not differ with gender. Based on these parameters, we measured the average age of cardiomyocytes, their age distribution, turnover rate and time to acquire the senescent phenotype to define the biology of myocardial aging as a function of lifespan. In the female heart, myocyte turnover occurs at a rate of 10%, 15% and 40% per year at 20, 60 and 100 years of age, respectively. Corresponding values in the male heart are 7%, 12% and 32% per year, documenting that cardiomyogenesis involves a large and progressively increasing number of parenchymal cells with aging. In conclusion, the human heart is a highly dynamic organ in which progressive myocyte loss is at least in part counteracted by enhanced myocyte renewal. Myocyte regeneration in the physiologically aging heart takes place at previously unexpected levels in both women and men.
Abstract 190: Protein Phosphatase 2Ce Is a Novel Phospholamban Phosphatase that Exacerbates Cell Death and Suppresses Cardiac Contractility
Authors: Akaike Toru , Lu Gang , Wang Yibin , Ruan Hongmei
Published: July 2021

Regulation of sarcoplasmic reticulum (SR) calcium-ATPase (SERCA) activity is critical for calcium homeostasis in cardiomyocytes, and has a major impact on contractility and cellular viability of cardiomyocytes. The key regulators for SERCA activity include protein kinases, cAMP dependent protein kinase A and calcium/calmodulin dependent protein kinase II, and protein phosphatase 1. In this report, we have discovered that protein phosphatase 2Ce (PP2Ce) is a novel serine/threonine protein phosphatase specifically targeted to SR membrane in cardiomyocytes. PP2Ce was detected to interact with phosphlamban in heart. Recombinant PP2Ce protein showed a potent and specific activity towards the calcium/calmodulin dependent protein kinase II dependent phospholamban phosphorylation at threonin 17 site with no significant activity to cAMP dependent protein kinase A dependent phospholamban phosphorylation at serine 16 site. Expression of PP2Ce blunted β-adrenergic stimulated increase of phospholamban phosphorylation without affecting phosphorylation of ryanodine recepter or troponin I. PP2Ce expression reduced β-adrenergic stimulated intracellular calcium transient in isolated adult rabbit ventricular myocytes, and promoted hydrogen peroxide induced cell death in cultured neonatal rat ventricular myocytes. Transgenic mice with cardiac specific expression of PP2Ce showed no significant basal phenotype. However, in isolated perfusion heart preparation, β-adrenergic stimulated contractility was significant reduced in PP2Ce transgenic hearts comparing to wild type controls. Furthermore, we observed significantly larger infarct sizes and more impaired functional recovery following global ischemia/reperfusion injury in the transgenic hearts comparing to wild type controls. Therefore, PP2Ce is a novel component of SR calcium regulatory network that has a potentially important role in cell death regulation and cardiac contractility.
Abstract 191: Functional Dissection of Myosin Binding Protein-C Phosphorylation
Authors: Gupta Manish K , Gulick James , Osinska Hanna , James Jeanne , Lasko Valerie , Nieman Michelle , Lorenz John N , Robbins Jeffrey
Published: July 2021

Rationale:
Phosphorylation of cardiac myosin binding protein C (cMyBP-C) plays an important role in cardiac function and is actively regulated both in the normal heart and during the development of cardiac disease. Hence it is critical to understand the mechanistic and functional consequences of cMyBP-C phosphorylation

Objective:
To examine the non-equivalency of the group of three phosphorylatable sites (Ser-273, Ser-282, and Ser-302) whose phosphorylation states have been defined as being essential when ablated in concert in a transgenic mouse model. However, the distinct role, if any, of each individual phosphorylation site is not established.

Methods and Results:
Three transgenic lines were generated: DAA that expressed cMyBP-C containing Asp273-Ala282-Ala302, AAD, in which a charged amino acid was placed at residue 302 (Ala273-Ala282-Asp302) and SDS Ala273-Asp 282-Ala302). The effects of these replacements were determined at the whole animal and organ levels. The DAA and AAD mice showed pathology that was more severe than either the cMyBP-C null animals or animals that expressed cMyBP-C that was unable to be phosphorylated at any of the three sites. Both the DAA and AAD hearts exhibited atrial enlargement, left ventricular chamber dilation, extensive interstitial fibrosis, irregular cardiac rhythm and sudden cardiac death. Ultrastructural studies of DAA mice confirmed myocyte disarray. Yeast two hybrid analyses were used to determine the effects of altering the charge at these sites with respect to the ability of cMyBP-C to bind to the S2 domain of myosin or cardiac actin. While phosphorylation at either Ser-273 or Ser302 did not affect myosin S2 interaction, substitution of Ser 282 with a charged residue prevented interaction of cMyBP-C with both S2 myosin and actin.

Conclusion:
Our results confirm the non-equivalency of these three sites in terms of the effects of their post-translational modifications on cMyBP-C functionality in the heart. Ser-282 appears to be the key residue in controlling S2 interaction with the protein. Chronic phosphorylation at one of these sites in the absence of the other, adjacent sites' ability to be phosphorylated leads to severe cardiac remodeling and sudden death.
Abstract 193: Knockdown of Nucleosome Assembly Protein 1-Like 1 Promotes Dimethyl Sulfoxide-Induced Differentiation of P19CL6 Cells into Cardiomyocytes
Authors: Gong Hui , Li Lu , Yan Yuan , Xue Yuanyuan , Chen Zhidan , Jiang Guoliang , Zhang Guoping , Yang Pengyuan , Zou Yunzeng
Published: July 2021

Various stem cell populations have the potential for cardiac repair and regeneration after transplanted into ischaemic injury heart. However, the low generation of new cardiomyocytes limits the clinic outcomes of stem cell therapy to a great extent. To develop novel strategies for enhancement the efficacy of stem cell differentiation into cardiomyocytes, we employed iTRAQ-coupled 2-D LC-MS/MS proteomics technology workflow to identify novel proteins which could promote P19CL6 cells differentiation into cardiomyocytes induced by dimethyl sulfoxide dimethyl sulfoxide (DMSO). 213 differentially expressed proteins were identified during differentiation of P19CL6 cells into cardiomyocytes. Nine proteins, Nap1L1, Sub1, Hspa5, Ywhaz, Sod1, Prdx2, Mif, Anp32a and Eef2, were confirmed by the real-time polymerase chain reaction (PCR) and western blotting respectively. iTRAQ ratios showed the down-regulation of nucleosome assembly protein 1-like 1 (Nap1L1) during the differentiation of P19CL6 cells into cardiomyocytes, which was confirmed by real-time PCR and western blot results. Further study indicated that knockdown of Nap1L1 expression promoted DMSO-induced cardiomyocytes differentiation in P19CL6 cells characterized by the increased expresison of cardiac specilal transcription factors (GATA4, MEF2C) and proteins (cTNT, ANP and BNP). This work suggested that Nap1L1 play an important role in DMSO-induced cardiomyocytes differentiation of P19CL6 cells. It also provides new insights and platforms for stem cell therapy in cardiovascular disease.
Abstract 197: Plasminogen Is Required for Hematopoietic Stem Cell-Mediated Cardiac Repair After Myocardial Infarction
Authors: Gong Yanqing , Li Ying , Hoover-Plow Jane
Published: July 2021

Myocardial infarction (MI) is the primary cause of death throughout the United States. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize hematopoietic progenitor and stem cells (HPSC) to improve cardiac recovery after MI. However, poor-mobilization to G-CSF is observed in 25% of patients and 10-20% of healthy donors. Therefore, a better understanding of the underlying mechanisms may offer novel approaches for G-CSF-mediated therapeutics. Our previous studies have identified an essential role of Plg in HPSC mobilization from bone marrow (BM) in response to G-CSF. Here, we investigate the role of Plg in G-CSF-stimulated cardiac repair after MI. Our data show that G-CSF significantly improves cardiac repair including increasing neovascularization in the infarct area, and improving ejection fraction and LV internal diameter determined by echocardiogram in WT mice. No improvement on heart function is observed in Plg
-/-
mice, indicating Plg is required for G-CSF-regulated cardiac repair after MI. To investigate whether Plg regulates HPSC recruitment to the ischemic area, BM transplantation with EGFP-expressing BM cells was performed to visualize BM-derived stem cells in infarcted tissue. Our data show that G-CSF dramatically increases recruitment of GFP
+
cKit
+
cells (by 12 fold) in WT mice but not in Plg
-/-
mice. In addition, BM stem cell-derived vessels and arteries are infrequent in Plg
-/-
mice suggesting that Plg enhances cardiac repair by promoting stem cell recruitment to the lesion. In further studies, we investigated the role of Plg in the regulation of SDF-1/CXCR-4 axis, a major regulator for HPSC recruitment. Our results show that G-CSF significantly increases CXCR-4 expression in the infarcted area in WT mice. While G-CSF-induced CXCR-4 expression is markedly decreased (80%) in Plg
-/-
mice, suggesting Plg may regulate CXCR-4 expression during HSPC recruitment to injured heart. Interestingly, Plg does not affect SDF-1 expression in response to G-CSF treatment. Taken together, our findings have identified a critical role of Plg in HSPC recruitment to the lesion site and subsequent tissue repair after MI. Thus, targeting Plg may offer a new therapeutic strategy to improve G-CSF-mediated cardiac repair after MI.
Abstract 198: Angiotensin II Inhibits Cardiac Angiogenesis via the Cooperation of p53 and Jagged 1
Authors: Guan Aili , Gong Hui , Ye Yong , Jia Jianguo , Zhang Guoping , Sun Aijun , Ge Junbo , Zou Yunzeng
Published: July 2021

It is well established that angiotension II (Ang II) is an important regulator in vascular homeostasis. Under certain conditions, Ang II could exert anti-angiogenic effects in cardiovascular system. However, the potential mechanism is unclear. P53 has been reported to suppress angiogenesis by promoting hypoxia-inducible factor-1 (Hif-1α) degradation. This study was conducted to determine the contribution of P53 and the underlying mechanism to the anti-angiogenic effect of Ang II. Angiogenesis was determined by tube formation from the cardiac microvascular endothelial cells (ECs). Microvessel density and cardiac function were analyzed in mice subjected to Ang II infusion (200 ng/kg/min ) or vehicle for 2 weeks. Ang II (1μM) greatly inhibited tube formation and stimulated phosphorylation and upregulation of P53 in cultured cardiac ECs. P53 inhibitor, pifithrin-α (PFT-α,3.0mg/kg), significantly reversed the inhibitory effect of Ang II on tube formation. Vascular endothelial growth factor (VEGF ) and Hif-1α has been reported as important pro-angiogenetic factors. The present study indicated that Ang II decreased VEGF concentration in cultured medium and downregulated Hif-1α expression in cultured ECs. Interestingly, Ang II also stimulated the upregulation of Jagged 1, a ligand of Notch, but it didn't affect the Delta-like 4 (Dll 4) , another ligand of Notch, expression in cardiac ECs. However, PFT-α partly abolished these effects of Ang II. These results were consistent with the study
in vivo.
Further research revealed that siRNA-Jagged 1 transfection in cultured ECs dramatically abolished the phosphorylation of P53 and the downregulation of Hif-1α induced by Ang II. Additionally, Ang II- induced inhibitory effect on capillary formation was blocked by siRNA-Jagged 1 transfection in cultured cardiac ECs. In conclusion, Ang II promoted the phosphorylation and upregulation of P53, and increased Jagged 1 expression, the upregulation of Jagged 1 in turn stimulated the phosphorylation of P53, which resulted in the downregulation of Hif-1α and VEGF, then induced the inhibitory effects of Ang II on capillary formation. The present data suggest that Ang II exerts anti-angiogenesis via the cooperation of P53 and Jagged 1
in vitro and in vivo.
Abstract 20: Resident Cardiac Stem Cell Growth Determines the Number of Ventricular Cardiomyocytes in the Mouse Heart
Authors: Ogórek Barbara , Ferreira-Martins João , Cappetta Donato , Matsuda Alex , Signore Sergio , D'Amario Domenico , Kostyla James , Steadman Elisabeth , Caballero Virginia , Ide-Iwata Noriko , Sanada Fumihiro , Hosoda Toru , Anversa Piero , Leri Annarosa , Rota Marcello , Kajstura Jan
Published: July 2021

The objective of this study was to determine the role of c-kit-positive cardiac stem cells (CSCs) in the formation of the heart during prenatal life, and immediately after birth. Mice in which EGFP is under the control of the c-kit-promoter were employed to measure the number of CSCs (Ns), the fraction of cycling MCM5-positive CSCs (f) and the length of the cell cycle (Ts) in CSCs. The number of CSCs committed to the myocyte lineage (LCC: lineage committed cells) included myocyte progenitors (c-kit-positive, Nkx2.5-positive cells), myocyte precursors (c-kit-positive, Nkx2.5-positive, and α-sarcomeric actin-positive cells) and replicating amplifying myocytes (c-kit-negative, Nkx2.5-positive, α-sarcomeric actin-positive, and MCM5-positive cells). These variables derived from CSC growth and lineage specification were evaluated to define the rate of formation of terminally differentiated myocytes (r). Based on a hierarchically structured cell organization, the rate of entry (Rs) of CSCs into the cell cycle was computed from Rs = f x (Ns/Ts), and the rate of generation of mature myocytes, r, was obtained from r = Rs x 2
Gt
= ((f x Ns)/Ts) x 2
Gt
. The exponent Gt defines the number of transit generations, i.e., the number of divisions that one CSC has to go through before it acquires the terminally differentiated myocyte phenotype. To validate this scenario and establish the number of post-mitotic myocytes formed, the primary data listed above were collected at E9, E14, E19 and P1. The number of mature cardiomyocytes generated by 1 CSC in 1 day was 1.1 x 10
3
, 20 x 10
3
, 501 x 10
3
, and 440 x 10
3
at E9, E14, E19 and P1, respectively. The total number of myocytes (Nm) formed from E9 to E14, E19 and P1 was derived from an exponential equation with the best fit to the experimental data: Nm = exp (0.69 x t) where Nm is the number of myocytes and t is time in days. Accordingly, CSCs generated 1 x 10
5
, 1 x 10
6
and 1.8 x 10
6
myocytes at from E9 to E14, E19 and P1, respectively. These values accounted for all parenchymal cells present at mid and late gestation and in the neonatal heart measured morphometrically. Thus, the expansion of the myocyte mass during embryonic, fetal and immediate postnatal development is controlled by activation, growth and differentiation of resident c-kit-positive CSCs.