bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 113: Mast Cell Plays Pivotal Role in Nicotine-Induced Atherosclerotic Plaque Progress and Instability
Authors: Chen Han , Wang Chen , Xu Yinchuan , Hu Xinyang , Wang Jian-an
Published: July 2021

Objectives:
Nicotine has been identified to promote atherosclerosis. But the mechanism of nicotine induced atherogenesis has not been well elucidated. This study focus on the role of mast cell in nicotine induced atherogenesis and plaque instability.

Methods:
Peritoneal administration of 100mM disodium cromoglicate (DSCG) was introduced to inhibit mast cell degranulation. 45 ApoE deficient mice were divided into 3 groups: high-fat diet, high-fat diet + nicotine, and high-fat diet + nicotine + DSCG. After 12 weeks of treatments, atherosclerotic lesion size of the aortas were quantified. Toluidine blue and tryptase staining identified mast cell count and activation at the lesion. Immuno-staining were used to evaluate the inflammatory filtration, smooth muscle cell proliferation and collagen content in the lesion. In vitro , bone marrow-derived mast cells (BMMCs) were harvested and treated with PBS as a negative control, compound 48/80 as a positive control,100μg/ml nicotine, nicotine with 100mM DSCG pretreatment and nicotine with 10μg/ml mecamylamine pretreatment. At 0.5hr,1hr and 2hrs, supernatants were harvested to analyze the mast cell degranulation. Futhermore, conditioned medium were also used to induce the macrophage migration and foam cell formation.

Results:
Nicotine increases plaque size, and macrophage infiltration, decreases smooth muscle collagen content along with the increases in mast cells count and activation ratio at the lesion, which could be inhibited by DSCG.Nicotine induced mast cell degranulation at 2 hours comparing to PBS (43.60% vs 2.3%) , which could be inhibited by mast cell stablizer DSCG (23.7%) and nAChR blocker mecamylamine (20.35%). Macrophage migration ability in the compound 48/80 and nicotine conditional medium group were significantly higher comparing to PBS, DSCG and mecamylamine group. Foam cell formation ratio in the compound 48/80 and nicotine conditional group were significantly higher comparing to PBS, DSCG and mecamylamine group.

Conclusions:
Nicotine induces mast cell degranulation through nAChR and then increases macrophages function, which leads to plaque instability. Administration of mast cell stabilizer showed potential of preventing nicotine induced atherogenesis.
Abstract 124: Parthenogenetic Stem Cell-derived Cardiomyocytes Express Major Histocompatibility Complex-I only after Inflammatory Stimulation
Authors: Galla Satish , Didie Michael , Muppala Vijayakumar , Dressel Ralf , Zimmermann Wolfram Hubertus
Published: July 2021

Background:
Pluripotent parthenogenetic stem cells (PSCs) can be directed towards a cardiac fate and utilized in tissue engineered heart repair. In vivo applications of tissue engineered allografts are compromised by expression of mismatching major histocompatibility complex proteins (MHC; encoded in the murine H2 locus). Here we investigated whether PSC-derived cardiomyocytes (CM) express MHC-I.

Methods:
Mouse PSCs (A3-line from B6D2F1 strain with haploidentical H2K
d
-locus) expressing a CM-specific neomycin-resistance and GFP were differentiated and purified for CM by addition of G418 (85% purity by FACS for actinin). To simulate heart muscle biology in vitro, we made use of engineered heart muscle (EHM) constructed from PSC-derived CM (75%), growth-inhibited murine embryonic fibroblasts (MEF (25%); NMRI mice), and collagen type I. MHC class-I H2K
d
(MHC-I) expression was assessed on CM and Non myocytes before EHM assembly and from enzymatically digested EHMs (cultured for 10 days) by FACS. Interferon gamma (IFNγ) was added for 48 h to stimulate MHC-I expression. As a reference, we investigated MHC-I expression in CM from neonatal mice and adult mouse hearts by FACS and by immunofluorescence staining.

Results:
EHM showed a positive ionotropic response to beta-adrenergic stimulation which could be reduced by muscarinergic stimulation. A3-CM, in contrast to Non myocytes, showed negligible expression of MHC-I (1±0.5% vs. 60±10% positive cells; n=3). EHM culture did not change MHC-I expression in CM. IFNγ treatment resulted in a marked increase of MHC-I-expression in CM monolayer culture (40±6%; n=3) and in EHM (30±8%; n=3). For comparison, 30% (n=2) neonatal CM expressed MHC-I while MHC-I was not detectable in adult CM.

Conclusion:
PSC-derived CM show a similarly low expression of MHC-I as adult CM and respond with MHC-I upregulation to IFNγ stimulation. This suggests a mature immunological response in PSC-CM with important implications for in vivo applications, i.e., MHC-I matching will likely be a prerequisite for successful allografting of PSC-EHM.
Abstract 126: Pregnancy Hormones Induce An Increase Of Capillary Density In The Mouse Heart
Authors: Hesse Michael , Korzus Daniel , Thaben Kristina , Fleischmann Bernd K
Published: July 2021

During pregnancy the maternal organism undergoes a variety of adaptive physiological changes. The cardiovascular system is strongly affected and its response is characterized by cardiac hypertrophy. The molecular mechanisms underlying these adaptations are still poorly understood. The recent postulation of resident organotypic stem cells in the cardiovascular system prompted us to explore the potential involvement of these cells and the related signaling cascades involved in this adaptation process in the heart.

We have analyzed cell proliferation using immunostainings in mouse hearts at different stages during pregnancy. The average percentage of proliferating cells in cardiac sections increased from virgin controls to gestational day 3 (GD3), peaked at GD14, and immediately stopped after delivery. Co-staining revealed that the majority of proliferating cells were fibroblasts and endothelial cells (ECs). The lack of cardiomyocyte proliferation was corroborated using BrdU pulse chase experiments analyzed in single cells upon Langendorff perfusion. The proliferation of fibroblasts and ECs reflects angiogenesis, which was underscored by an increase of capillary density, and extracellular matrix remodeling known to occur during pregnancy induced hypertrophy. To mimic the action of pregnancy hormones we implanted pellets with constant release of hormone for 21 days subcutaneously in ovariectomized mice. Capillary density was significantly increased in mice receiving progesterone-pellets alone or in combination with estrogen-pellets without induction of hypertrophy. Thus, increase of capillary density is directly induced by pregnancy hormones. Assessment of differentially expressed genes after hormone treatment revealed induction of angiogenetic factors and their receptors.

We are currently investigating the mechanisms underlying the pregnancy hormone-induced proliferation in cardiac ECs and fibroblasts.
Abstract 132: Cardiomyocyte Specific Conditional Overexpression Of Stromal Cell Derived Factor 1 Facilitates Cardiac Regeneration After Permanent Coronary Artery Ligation In Mice.
Authors: Obal Detlef , Brittian Kenneth , Book Michael , Bhatnagar Aruni , Guo Yiru , Bolli Roberto , Rokosh Gregg
Published: July 2021

Background:
Interruption of cardiac stromal cell derived factor 1 (SDF1)-CXCR4 axis by chronic AMD3100 administration increased myocardial injury after permanent coronary artery ligation demonstrating the important role of this chemokine in cardiac regeneration.

Hypothesis:
Cardiomyocyte specific conditional overexpression of SDF1 prevents heart failure after permanent coronary ligation and facilitates cardiac regeneration.

Methods and Results:
Tetracycline-controlled, αMyHC promoter directed overexpression of cardiac SDF1, resulted in a significant increase of SDF1 expression (SDF1: 8.1 ng/mg protein) compared to littermate WT mice (0.02 ng/mg protein) four weeks after doxycycline withdraw. SDF1 overexpression increased AKT and casein kinase 1 levels in the heart. Although there was no difference in cardiac function and scar size 1 week after infarction, SDF1 overexpression improved left ventricular (LV) ejection fraction (SDF1 [n=13]: 47±5% [mean±SEM] vs. WT [n=15]: 29±4%, p<0.05) decreased end-diastolic volume (78±10 vs. 158±30, p<0.05) and reduced infarct size measured by trichrome staining (13±3% vs. 23±3% of LV wall, p<0.05) 4 weeks after permanent ligation. Bromodeoxyuridine (BrdU) staining revealed increased regeneration indicated by a 5-fold increase in BrdU
+
cardiomyocyte (CM) nuclei in the borderzone of the infarct (22±3% vs. 5±1% CM nuclei, p<0.01). Increased proliferation in SDF1 mice was confirmed by a higher number of KI67
+
cells compared to WT mice. Cardiomyocyte cross sectional area in the border zone was significantly reduced in SDF1 mice (365±13 μm
2
vs. 434±10 μm
2
, p<0.001) while capillary density was unchanged (2348±151/ mm
2
vs. 2498±153/ mm
2
) compared to WT mice.

Conclusion:
This study demonstrates for the first time that cardiac specific overexpression of SDF1 increases myocardial regeneration and improves LV function 4 weeks after permanent coronary ligation.
Abstract 138: The Use of β-1.3/1.6-D-Glucan as Complementary Therapy for Cardiovascular Diseases
Authors: Sugita Peter
Published: July 2021

Cardiovascular disease nowadays is still the one silent killer over the world.So far, current standard therapies for cardiovascular diseases do not reach expected results such as the improving heart function by rehabilitating the damaged cardiomyocytes

For thousands of years
Ganoderma lucidum
extract has been used as herbal therapy for various types of diseases, including cardiovascular diseases. An International Publication No. WO 2005/084692 A2 with the title
Effect of Glucan on Stem Cell Recruitment and Tissue Repair
mentions that “
the orally administered glucan enhances proliferation, activation and differentiation of committed stem progenitor cells by functioning with the complement system by providing a second signal for CR3 activation
.” Furthermore, it is also discussed that “
the committed stem cells are selected from the group consisting of committed stem cells from the liver, heart, muscle, kidney and neural tissue
.”

In 1995 we began a research on β-D-Glucan derived from the purified extract of East-Java Indonesian
Ganoderma lucidum
mycelia.At the end of 2009 we have the first clinical experience the use of our β-1.3/1.6-D-Glucan for cardiovascular diseases patient. A bypass surgery patient who was also a candidate for heart transplant experienced a good clinical result after 8 months use of our beta glucan with dose of 3 x 200 mg beta glucan a day. A significant improvement of Ejection Fraction (EF) was reached from 22% to 44%. Previously, when he only took standard drug therapy for cardiovascular disease after bypass surgery, the improvement of EF was not significant (from 13% as baseline to 22%).This result also applied to 4 other patients.

We presumed that this specific
Ganoderma lucidum
β-D-Glucan has similar mechanism of actions as mentioned in the above publication and also in a journal
Modulating Toll-like receptor mediated signaling by(1-3)-β-D-glucan rapidly induces cardioprotection
.

The conclusion is β-D-Glucan could serve as a complementary therapy in cardiovascular diseases but it still needs further investigations through in vitro / in vivo assays and clinical trials.
Abstract 14: The Cytoskeletal Architecture of Myocytes Derived from Human Cardiac Progenitor Cells
Authors: Pasqualini Francesco , Di Sante Moises , Pereira João D , Anversa Piero , Rota Marcello , Leri Annarosa
Published: July 2021

The stem cell antigen c-kit characterizes a heterogeneous pool of human cardiac progenitor cells (hCPCs) that exhibit a remarkable degree of regenerative potential and are currently employed in clinical trials. While this hCPC pool contains distinct subpopulations of c-kit+ cells that preferentially differentiate into muscular or vascular cardiac cells, we hypothesize that hCPCs may be coerced to specify only along the cardiomyogenic lineage by manipulating the Wnt/β-catenin pathway.

We report that pharmacological inhibition of the non-canonical Wnt pathway facilitated the commitment of more than >95% c-kit+ hCPCs to the cardiomyocyte lineage after 4 days in-vitro: this constitutes a substantially more homogeneous population than previously reported with dexamethasone treatment.

The hCPC-derived myocytes stained positive for Nkx2.5, a transcription factor that orchestrates cardiomyogenic differentiation, and for the contractile protein sarcomeric α-actin. To test if we could push the cells towards a more mature phenotype, we mimicked the cyclic modulation of the Wnt pathway observed during development. While activation of Wnt signaling resulted in widespread cell death and reduction in cell size, subsequent Wnt inhibition prompted the spared cells to proliferate. With this protocol, hCPC-derived myocytes increased in size and displayed more mature cytoskeletal architectures. In contrast with dexamethasone treated cells, where the localization of α-sarcomeric actinin is mostly diffuse in the cytoplasm, here we observed both Z-bodies and Z-disks like structures. The latter exhibited a periodicity of ~1.6 um and were clustered in larger, more aligned actin bundles. This finding suggests that the tension developed along these cytoskeletal components may play a role in the recruitment of sarcomeric proteins.

In conclusion, Wnt signaling inhibition in hCPCs may be sufficient to obtain a homogeneous population of cells with features of myocytes, characterized by improved cytoskeletal organization than dexamethasone treated cells and similar to that observed in myocytes derived from human induced pluripotent stem cells.
Abstract 142: Defining the Non-Myocyte Compartment is Key for Enhanced Maturation of Human Engineered Heart Muscle
Authors: Tiburcy Malte , Hudson James E , Ziebolz Dirk , Zimmermann Wolfram H
Published: July 2021

Background:
Tissue engineering of heart muscle from human pluripotent stem cells holds great potential for in vitro studies, disease modeling, and cardiac replacement therapy. A number of variables may however affect maturation and function of human cardiomyocytes (CM) in tissue engineered heart muscle (EHM). Here, we hypothesized that defined non-myocyte (NM) populations support structural and functional maturation of EHM.

Methods and Results:
To investigate the role of non-myocytes (NM) for heart muscle assembly in vitro we generated EHM from purified CM (93±1.5% actinin+) and a mixture of CM and NM (70/30%). Notably, only the NM-supplemented EHM generated measurable forces (0.8±0.1 mN, n=9) with anisotropically aligned cardiomyocytes. Depending on pluripotent stem cell line and differentiation protocol the NM compartment may vary considerably. To further define the influence of the NM compartment we generated EHM from HES2-derived CM with undefined NM, i.e the NM typically derived during cardiac differentiation, and defined NM (fibroblasts). Defined EHM were more mature with higher forces and lower variability between experimental series (defined: 9.8±0.9 nN/CM, undefined: 4.7±1.4 nN/CM, n=10/9), higher EC50 for calcium, and enhanced inotropic response to isoprenaline despite comparable CM:NM composition of 1:1. Increased actinin protein per CM, a reduction of MLC2V/2A double positive CM, and evidence of CM cycle withdrawal indicated enhanced ventricular maturation in defined EHM. Next, we tested whether defining cell composition and NM in iPS-derived EHM will yield a comparable functional phenotype to HES2-EHM. In agreement with the above data, defined iPS-EHM displayed advanced functional maturation with high specific forces, comparable calcium EC50, and inotropic response to isoprenaline.

Summary and Conclusions:
Here we demonstrate that defining the NM compartment is essential for optimized human heart muscle formation and maturation in vitro. Moreover, our data provide (1) evidence for the applicability of EHM in modelling of heart muscle development and (2) a strong rationale for the need to define CM and NM compartments in tissue engineered myocardium to reduce variability in applications such as disease modelling.
Abstract 144: Post-myocardial Infarction Therapeutics Using Cardiovascular Progenitor Cells (PC) Derived from Induced Pluripotent Stem Cells (iPSC)
Authors: Wang Yigang
Published: July 2021

Objective:
We sought to assess the cardiac protective effects after MI of (1) PC differentiated directly into cardiomyocytes (CM) and endothelial cells (EC) to the site of injury, or (2) paracrine factors released from PC.

Methods:
These concepts were evaluated by using iPSC-derived PC genetically modified to express the herpes simplex virus thymidine kinase (TK) under the control of cardiomyocyte (NCX1) or endothelial cell (VE-cadherin) specific promoters. PC expressing the TK permitted ablation at the first week or the third week by iv ganciclovir (GCV). If GCV applied at the first week, but not at the third week, altered cardiac function, we would conclude that myocardial contractile recovery depends on CM and EC-derived from iPSC. If the beneficial effects on cardiac function persisted after GCV was given at the third week, we would surmise that the PC effect was via by a paracrine action. MI created by ligation of LAD, the cell patch with PC was applied to the scarred myocardium. Rats were treated with GCV at 1 or 3 weeks to ablate implanted PC. Echocardiography, vessel density, and histological analysis were used to obtain endpoints for this study.

Result:
In vivo
: The levels of IGF-1α and VEGF released from ischemic tissues were significant higher in the cell patch group. Heart function, infarction size, and vessel density were significantly improved after cell patch treatment. However, this beneficial effect on cardiac function was completely abolished in the group given GCV at week 1, but only partially abolished in the group given GCV at week 3 compared to the untreated cell patch group.

Conclusions:
Taken together, these data support our conclusion that iPSC-derived cardiovascular lineages (CM and EC) contribute directly to an improved cardiac performance and attenuated remodeling, and that paracrine factors also play a supporting role in the restoration of heart function after MI.
Abstract 156: Involvement of β-adrenergic System in Myocardial Dysfunction Induced by Obesity
Authors: Lima-Leopoldo Ana Paula , Ferron Artur , Jacobsen Bruno , Campos Dijon , Luvizotto Renata , Cunha Márcia R , Cicogna Antonio , Leopoldo Andre
Published: July 2021

Several structural and functional changes of the heart have often been associated with human and experimental models of obesity. Some factors have been suggested as responsible for possible cardiac abnormalities in models of obesity, among them β-adrenergic system, an important mechanism of regulation of myocardial contraction and relaxation. The objetive of present study was to evaluate the . Thirty-day-old male Wistar rats were assigned to one of two groups: control (C) and obese (Ob). The C group was fed a standard diet and Ob group was fed cycles of four unsaturated high-fat diets for 15 weeks. The body fat was measured from the sum of the individual fat pad weights and the obesity was defined by adiposity index. Isolated papillary muscle preparation was performed under basal conditions and after inotropic and lusitropic maneuvers. β-adrenergic system was evaluated by using cumulative concentrations of isoproterenol and Western Blot. After 15 weeks, the Ob rats developed higher adiposity index than C rats. Obesity promoted comorbities such as glucose intolerance, insulin resistance, hyperleptinemia, and dyslipidemia; however, were not associated with changes in systolic blood pressure. The cardiac structure results post-death showed that obesity caused cardiac hypertrophy. Furthermore, Ob muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus and increased extracellular Ca2+ was compromised. There were no changes in cardiac function between groups after β-adrenergic stimulation. The obesity was not accompanied by changes in protein expression of Gsα, β1 and β2 adrenergic receptors. In conclusion, the myorcardial dysfunction caused by unsaturated high-fat diet-induced obesity, after 15 weeks, is not related to β-adrenergic system impairment.
Abstract 162: Single Nucleotide Polymorphism of the B-Type Natriuretic Peptide Helps Predict the Presence of Significant Coronary Artery Disease
Authors: Li Terry Y , Tse M. Y , Ventura Nicole M , Hetu Marie-France , Johri Amer M , Pang Stephen C
Published: July 2021

Early detection and diagnosis of coronary artery disease (CAD) is crucial in reducing the morbidity and mortality. The clinical standard for detecting CAD is by angiography which is associated with rare but important clinical risks. Recent studies have shown that up to 40% of patients referred for angiography do not have significant disease. This discrepancy between referral and diagnosis may be improved by the utilization of genetic screening. A single nucleotide polymorphisms (SNP) of the B-type natriuretic peptide gene (NPPB), rs198389, was previously found to be associated with several cardiovascular diseases. Our objective was to determine whether detection of genetic variation could contribute to better selection of patients referred for angiography. We hypothesized that an SNP analysis of the NPPB gene may help differentiate patients with significant disease from those with non-significant CAD. Ninety-three patients referred for coronary angiography at the Kingston General Hospital Cardiac Catheterization Lab were consented for genetic screening. Blood samples were collected during angiography procedure. Genomic DNA was isolated from leukocytes, and screened for SNPs using a real-time PCR-based TaqMan SNP Assay. We found that more males were referred for coronary angiography than females: 69% versus 31%. Two out of ten males (20%) were found to have no or minimal CAD. In contrast, 48% of females were found to have non-significant CAD. Older age (≥ 69 years) was deemed to be a significant predictor of CAD in the total recruited population (odds ratio of 3.4), but no age difference was found between healthy and diseased females. Additionally, a mutation of the B-type natriuretic peptide gene (NPPB) was found to be a significant predictor of CAD in the younger population (< 69 years), with an odds ratio of 5.9. We found a significant difference between patterns of CAD development in males and females, suggesting that different diagnostic criteria should be used depending upon gender. Moreover, younger individuals with two copies of the major allele for the NPPB SNP were more likely to develop CAD, making this SNP a potential factor in the prediction of CAD in younger population.
Abstract 172: Lumican-null Mice Are Susceptible to Aging and Isoproterenol-induced Myocardial Fibrosis
Authors: Chu Pao-Hsien
Published: July 2021

Aims:
With aging and stresses, the myocardium undergoes structural remodeling and often leading to fibrosis.

Main Methods:
To examine whether lumican, one of the class II small leucine-rich proteoglycans, has a role in cardiac remodeling and fibrosis, we analyzed the basic cardiac phenotypes of lumican-null (Lum-/-) mice in both youth and elder, and then used the isoproterenol-induced cardiac fibrosis model to study the roles of extra-cellular matrix and apoptosis in cardiac remodeling.

Key Findings:
Higher mortality resulted from significantly impaired systolic function, and an increase of atrial natriuretic peptide secreted by the ventricles in response to excessive stretching of myocytes of Lum-/- mice in comparison to wild type littermates. In addition, Lum-/- mice exhibited higher level of TGF-β, collagen I/III, and membrane-type matrix metalloproteinase-1 (MT1-MMP, or MMP-14) during cardiac remodeling.

Significance:
Our data implicates that the lumican protein plays an important role in the pathogenesis of cardiac fibrosis.
Abstract 177: Promotion Of Nitroso-redox Balance By Beta 3 Adrenoceptor Agonism: Therapeutic Implications For Cardiovascular Complications Of Diabetes
Authors: Karimi Galougahi Keyvan , Liu Chia-chi , Garcia Alvaro , Fry Natasha A , Hawkins Clare L , Rasmussen Helge H , Figtree Gemma A
Published: July 2021

Rationale:
Disrupted balance between NO and O2.- is central in pathobiology of diabetes-induced cardiomyopathy and vascular dysfunction. We examined if stimulation of β3 adrenergic receptors (β3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re-establish NO/O2.- balance, relieve oxidative inhibition of key caveolar proteins and protect against diabetes-induced cardiovascular dysfunction.

Methods/Results:
A hyperglycemic, hyperinsulinemic state was established in male White New Zealand rabbits by infusion of the insulin receptor antagonist S961 (12 μg/kg/h). Diabetes induced NADPH oxidase-dependent glutathionylation (GSS-) of the caveolar proteins Na+-K+ pump’s β1 subunit and eNOS in cardiac myocytes and aorta, an oxidative modification that inhibits the pump and uncouples eNOS. Consistent with this, diabetes was associated with reduced electrogenic Na+-K+ pump current in voltage-clamped cardiac myocytes and impaired endothelium-dependent vasorelaxation. Selective β3 AR agonist CL316243 (CL, 40 μg/kg/h) restored NO levels analysed by spin-trapping of NO-Fe(DETC)2 complexes; decreased diabetes-induced elevation in O2.- measured by HPLC analysis of dihydroethidium oxidation products, improved endothelium-dependent vasorelaxation, and restored the Na+-K+ pump function in cardiac myocytes. These effects were mediated by CL abolishing diabetes-induced increase in eNOS-GSS and β1-GSS through a decrease in forward reaction rate for glutathionylation by suppressing diabetes-induced NADPH oxidase activation, which was further amplified by promotion of de-glutathionylation via enhancement in association of glutaredoxine-1, the enzyme catalysing de-glutathionylation, with eNOS and Na+-K+ pump.

Conclusion:
β3 AR activation re-established nitroso-redox balance and relieved oxidative inhibition of key caveolar proteins in diabetes. β3 AR agonists are promising in treatment of diabetes-induced cardiovascular complications.
Abstract 18: Coxsackievirus B3 Escapes Infected Cardiac Stem Cells via Ejected Autophagosomes
Authors: Sin Jon , Mangale Vrushali , McIntyre Laura , Feuer Ralph , Gottlieb Roberta A
Published: July 2021

Many viruses have been shown to upregulate host autophagic processes during the course of infection. Originally this process was thought to be a response mechanism of the host cell in order to engulf viral particles and destroy them to prevent viral spread and host cell death. However, certain viruses have been shown to strategically hijack the autophagic pathway to evade host immunity and increase viral replication. Coxsackievirus B (CVB) is a non-enveloped picornavirus that most commonly causes a self-limited febrile illness in young children, but in rare severe cases can progress to myocarditis, pancreatitis, or meningo-encephalitis. It has also been associated with late-onset idiopathic dilated cardiomyopathy. CVB type 3 (CVB3) has previously been documented to upregulate host autophagic machinery upon infection, and we report here that the virus can infect cardiac stem cells and other muscle progenitors and allow itself to be engulfed in autophagosomes which later get shed from the surface of the host cell. This is a novel method of viral dissemination especially for a “naked” virus which would typically escape the infected host by triggering cytolysis. However, cytolytic viruses subsequently become exposed to neutralizing antibodies. The fact that CVB3 can hide itself in host membranes presents several fascinating possibilities. Not only could the ejected autophagosome mask the virus from adaptive immunity, but it may also bypass receptor-mediated endocytosis and infect cell-types that might lack CVB3 receptors. This mode of viral escape represents a unique evolutionary adaptation which obscures the line dividing enveloped and non-enveloped viruses.
Abstract 180: Synergistic Salutary Effects of HMG-CoA Reductase Inhibitor and Angiotensin II Receptor Blocker on Load-induced Heart Failure
Authors: Maejima Yasuhiro , Isobe Mitsuaki
Published: July 2021

We have shown previously that combined HMG-CoA reductase inhibitor (statin) and angiotensin II receptor blocker (ARB) therapy significantly improves both symptoms and left ventricular (LV) function over time in patients with heart failure (HF) by a clinical study [
HF-COSTAR
Trial]. We elucidated the mechanisms of combination therapy with the ARB (losartan, LOS) and long-acting and statin (simvastatin, SIM) for the treatment of load-induced heart failure. Salt-loaded Dahl salt-sensitive (DS) rats were treated with vehicle, LOS (5mg/kg/day), SIM (2mg/kg/day) and LOS + SIM for 16 weeks. LOS and SIM in combination improved LV dysfunction (ΔLV fractional shortening; LOS = 60%, SIM = 42%, LOS + SIM = 24%,
p
<0.05), limited LV hypertrophy (ΔLV septal thickness; LOS = −21%, SIM = −18%, LOS + SIM = −13%,
p
<0.05) and reduced cardiac fibrosis (ΔLV collagen density; LOS = −26%, SIM = −16%, LOS + SIM = −28%,
p
<0.05) more than LOS or SIM alone. Both Rho and matrix metalloprotease-9 (MMP-9) activity in LV tissue were increased in untreated DS rats, and LOS and SIM in combination decreased these changes more than did LOS and SIM monotherapies. We confirmed that the plasma level of Exp-3174 (E3174), a LOS metabolite and a potent inverse agonist of angiotensin II receptor type 1, was higher in rats treated with LOS and SIM in combination than in those treated with LOS alone (E3174/LOS ratio; LOS = 2.6 ± 0.3 vs. LOS + SIM = 3.2 ± 0.2,
p
<0.05). Next, to mimic the response of volume-overload heart failure
in vitro
, cultured neonatal rat cardiomyocytes (CMs) were cyclically stretched. Stretch-induced increased CM hypertrophy was suppressed by pretreatment with both SIM and E3174 more than by pretreatment with LOS, E3174, SIM, or LOS and SIM in combination. Mechanical stretch also induced activation of extracellular signal regulated kinase (ERK) and the stretch-induced ERK activation of CMs was also significantly suppressed by SIM + E3174. In conclusion, LOS and SIM had beneficial myocardial effects in rats with salt-sensitive hypertension, partly through promoting the accumulation of plasma E3174. SIM enhanced the myocardial protective effects of LOS through suppression of Rho and MMP-9 activity. Thus, a combination of ARB with statin has a promising potential as a therapeutic strategy for HF.
Abstract 190: Disruption of P90 Ribosomal S6 Kinase Binding 3 to Muscle A-kinase Anchoring Protein In Vivo Via Adeno-associated Virus Expression of a Competing Peptide Attenuates Pressure Overload-induced Cardiac Hypertrophy
Authors: PASSARIELLO CATHERINE L , Kritzer Michael D , Thakur Hrishikesh , Sanders Michael , Li Jinliang , Kapiloff Michael S
Published: July 2021

RATIONALE:
Cardiac myocyte hypertrophy is the main compensatory response to chronic stress in the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. RSK3 contains a unique N-terminal domain that mediates RSK3 binding to the muscle A-kinase anchoring protein (mAKAPβ) scaffold. We have previously published that disruption of RSK3-mAKAPβ complexes using a competing peptide inhibited the phenylephrine-induced hypertrophy of neonatal ventricular myocytes in vitro. In vivo, RSK3 gene deletion in mice attenuated the concentric cardiac hypertrophy induced by pressure overload. We hypothesize that RSK3 anchoring to mAKAPβ in myocytes is required for cardiac hypertrophy in vivo.

METHODS AND RESULTS:
Adeno-associated viruses (AAV) are gene therapy vectors in development for the treatment of human diseases owing to their nonpathogenic capability for transducing non-dividing cells and their long-term transgene expression. We have used a recombinant AAV2/9 vector to express a mAKAPβ RSK3-binding domain (RBD)-GFP fusion protein under the control of the cardiac myocyte-specific cardiac troponin T promoter. 3 day-old C57BL/6 mice were injected intraperitoneally with either AAV-RBD-GFP or AAV-GFP control virus. At 8 weeks of age mice were subjected to transverse aortic constriction to induce pressure overload (TAC) for two weeks. Cardiac hypertrophy was attenuated in mice injected with the AAV-RBD-GFP virus (biventricular weight indexed to tibial length (mg/mm): 7.7, 8.6, and 9.2 for AAV-RBD, AAV-GFP and non-injected TAC cohorts, respectively; p<0.05 vs. both controls). Echocardiography both corroborated the inhibition of hypertrophy and revealed no deleterious effect on cardiac function attributable to the AAV-RBD-GFP vector.

CONCLUSIONS:
Anchored RSK3 regulates pathologic myocyte growth. AAV can successfully deliver a competing peptide inhibiting pathological hypertrophy and should be investigated further as a prevention and/or treatment for heart failure.
Abstract 20: Manipulating PHD2 to Promote Efficacy of Stem Cell Therapy for Myocardial Infarction
Authors: Wang Wei Eric , Yang Dezhong , Li Liangpeng , Peng Yulan , Wang Wei , Ye Linda , Duan Dayue , Chen Xiongwen , Houser Steven R. , Zeng Chunyu
Published: July 2021

OBJECTIVE:
Stem cell transplantation has had modest success as a treatment for myocardial infarction (MI). One of the limitations is the poor stem cell survival and function in the diseased microenvironment. We studied whether and how prolyl hydroxylase domain protein 2 (PHD2), a cellular oxygen sensor, enhances stem cell cardioprotective effects after transplantation into infarcted hearts.

METHODS and RESULTS:
Both adipose-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BM-MSCs) were used. Stem cells were transduced with lentiviral short hairpin RNA to silence PHD2 (shPHD2) and intramyocardially injected into infarcted heart in mice. ADSCs reduced cardiomyocyte apoptosis, fibrosis, and infarct size (MI+ADSCs: 39.4±3.3%;MI+PBS: 48.4±4.5%) and improved cardiac function (MI+ADSCs: 43.8±5.6%; MI+PBS: 37.2±3.0%). shPHD2-ADSCs exerted significantly more protection (infarct size: 22.6±3.0%; LVEF: 67.3±6.8%; p<0.05 vs. MI+ADSCs). PHD2 silencing induced greater ADSC survival (survival rate at 7 days post-transplantation: shPHD2-ADSCs: 14.7±4.2% vs. ADSCs: 3.4±0.8%, p<0.05), which was abolished by HIF-1alpha silencing. No ADSC gave rise to cardiomyocyte and ADSCs induced cardioprotection was mainly induced by paracrine function. Conditioned medium from shPHD2-ADSCs decreased cardiomyocyte apoptosis. Insulin-like growth factor-1 (IGF-1) levels were 3.8 times higher in the conditioned medium of shPHD2-ADSCs than ADSCs, and depletion of IGF-1 attenuated the cardioprotective effects of shPHD2-ADSC-conditioned medium. NF-kappaB activation was induced by shPHD2 to stimulate IGF-1 secretion via binding to the IGF-1 gene promoter. A combination of HIF-1alpha silencing and IGF-1 neurtralization blocked the beneficial effects of shPHD2-ADSCs for MI. Similar findings were observed with BM-MSCs.

CONCLUSIONS:
PHD2 silencing promotes stem cell survival in infarcted hearts and enhances their paracrine function to protect cardiomyocytes. Its prosurvival effect on stem cells is HIF-1alpha dependent, while it enhances stem cell paracrine function through NF-kappaB-mediated IGF-1 upregulation. PHD2 silencing in stem cells may be a novel strategy for enhancing the effectiveness of stem cell therapy after MI.
Abstract 204: HAX-1: A Novel Regulator of Energetics and Oxidative Stress in the Heart
Authors: Lam Chi K , Zhao Wen , Cai Wenfeng , Liu Guansheng , Bidwell Phil , Gardner George , Adly George , Kranias Evangelia G
Published: July 2021

Sarcoplasmic reticulum (SR) calcium handling is central not only in the control of heart function during excitation-contraction coupling but also in mitochondrial energetics and apoptosis. Recent studies have identified the anti-apoptotic protein, HS-1 associated protein X-1 (HAX-1) as a novel regulator of SR calcium cycling. Although HAX-1 has been shown to localize to mitochondria in various tissues, we found out that it also localizes to SR through its interaction with phospholamban (PLN) in cardiac muscle. Acute or chronic overexpression of HAX-1 in cardiomyocytes promoted PLN inhibition on the calcium ATPase (SERCA) and decreased cardiomyocyte calcium kinetics and contractile parameters. Accordingly, ablation of HAX-1 significantly enhanced SERCA activity and calcium kinetics. Furthermore, the HAX-1/PLN interaction appeared to also regulate cardiomyocyte survival. Indeed, overexpression of HAX-1 and the associated depressed SR Ca-load attenuated endoplasmic reticulum stress induced apoptosis, as evidenced by reduction of both caspase-12 activation and pro-apoptotic transcription factor C/EBP homologous protein induction during ischemia/reperfusion injury. In addition, the depressed SR Ca-cycling by HAX-1 overexpression was associated with reduced mitochondrial Ca-load as reflected by: a) hyper-phosphorylation of pyruvate dehydrogenase (PDH) and decreases in its activity, to diminish ATP production consistent with the attenuated energetic demand in these hearts; and b) reduced levels of reactive oxygen species, indicating protection from oxidative damage and preserved mitochondrial integrity. These findings suggest that HAX-1 is a key regulator of Ca-cycling, apoptosis and energetics in the heart. Thus, decreases in HAX-1 levels, observed during ischemia/reperfusion injury, may contribute to the deteriorated function and progression to heart failure development.
Abstract 207: Unc-51-like Kinase 1 (ULK1) Plays A Crucial Role In Mitophagy In Cardiomyocytes
Authors: Saito Toshiro , Sadoshima Junichi
Published: July 2021

The mitochondrion is an essential organelle that supplies ATP in cardiomyocytes (CMs). However, damaged mitochondria are harmful via the production of reactive oxygen species and induction of apoptosis in pathological conditions. Therefore, quality of mitochondria should be controlled tightly through various mitochondrial quality control mechanisms. Mitochondrial autophagy (mitophagy) is considered an integral part of this mechanism, and recent investigations uncovered the role of PINK1 and Parkin in mitophagy. However, these observations were made under artificial conditions, such as over-expression of Parkin or treatment with CCCP, and thus the precise mechanism has not been fully elucidated in more pathophysiologically relevant conditions. Recent evidence suggests that mitophagy can take place independently of ATG7, a molecule essential for the conventional form of autophagy, and that this form of autophagy is ULK1-dependent. We investigated the role of ULK1 and ATG7 in mediating mitophagy using mitochondria-targeted Keima (Mito-Keima) in cultured rat neonatal CMs. Keima has a bimodal excitation spectrum peaking at 440 and 560 nm, corresponding to the neutral and acidic pH, respectively. In CMs transfected with Mito-Keima, the fluorescent dots with a high 560nm/440nm ratio represent the mitochondria incorporated into autolysosomes which indicate mitophagy. Here we report that ULK1 plays a more predominant role in glucose deprivation (GD) -induced mitophagy than ATG7. Control CMs exhibited 8.7±1.0 % of the area of high-ratio dots per cells after GD. Knockdown of ULK1 significantly reduced the area to 2.3±0.9 % in CMs after GD (p<0.01, vs sh-Control). The reduction was significantly greater in CMs with knockdown of ULK1 than that of ATG7 (7.0±1.6 %, p<0.05, sh-ULK1 vs sh-ATG7). In addition, knockdown of Beclin1 and Drp1 also significantly decreased the area of high-ratio dots (about 1.0 % and 0.5 %, respectively). Overexpression of ULK1 was sufficient to induce mitophagy without starvation, whereas that of ATG7 was not. These results suggest that ULK1, Beclin1 and Drp1 play an essential role in mediating GD-induced mitophagy in CMs.
Abstract 213: Dystrophic iPSC-derived Cardiomyocytes Have Mislocalization of eNOS and Increased Susceptibility to Cell Death which is Reversed by the Nitrate-like Properties of Nicorandil.
Authors: Strande Jennifer L , Reiter Melanie , Gastonguay Courtney , Afzal M. Zeeshan
Published: July 2021

Background:
Cardiomyopathy is a leading cause of death in Duchenne and Becker muscular dystrophy (D/BMD) patients and specific therapies are lacking. Dystrophin deficiency is associated with mislocalization of nitric oxide synthase (NOS) whose dysregulation contributes to the pathogenesis of dystrophic cardiomyopathy. We have modeled dystrophic cardiomyopathy using patient-specific induced pluripotent stem cells (iPSCs) and show that DMD- and BMD- iPSC derived cardiomyocytes have mislocalization of endothelial (eNOS) but not neuronal NOS (nNOS). In this study, we determined whether nicorandil, a drug with nitrate-like properties, protects against stress-induced dystrophic cardiomyocyte injury.

Methods/Results:
DMD-iPSC (exon 3-6 deletion), BMD-iPSC (exon 45-53 deletion) and non-dystrophic (N)-iPSC lines were differentiated into cardiomyocytes (iCMs) and matured for 35-38 days. Immunofluorescence revealed eNOS co-localized with dystrophin along the sarcolemma in N-iCMs but not in DMD- or BMD-iCMs. eNOS also co-localized with caveolin-1 in all groups. nNOS did not co-localize with dystrophin but with the ryanodine receptor 2 in all groups. Both DMD- and BMD-iCMs have increased cell death as determined by TUNEL + staining when subject to 2 hours of metabolic stress and 4 hours of recovery (DMD: 66±3%, BMD: 29±4% vs. 1.5±0.04%; n=3-6). This was associated with dissipation of the mitochondrial membrane potential as monitored by laser-scanning confocal microscopy (DMD: complete loss, BMD: 441±168 vs. N: 891±183 AU; n=3-5). Nicorandil pretreatment was able to restore mitochondrial membrane potential and decrease cell death. The protective effects of nicorandil were abolished by ODQ, a selective inhibitor of NO-sensitive guanylyl cyclase.

Conclusion:
Nicorandil protects against stress-induced cell death and preserves mitochondrial function in dystrophic cardiomyocytes through its nitrite-like properties. This suggests a potential therapeutic role of nicorandil in the treatment of dystrophic cardiomyopathy.
Abstract 22: CXCR4 Expression on Endogenous Bone Marrow Stem/Progenitor Cells is Crucial for Preservation of Myocardial Function After MI
Authors: Wysoczynski Marcin , Solanki Mitesh , Ponnapureddy Rakesh , Gadde Rakesh , Bolli Roberto , Rokosh Gregg
Published: July 2021

Background:
Studies examining the role of bone marrow CXCR4 in the response to myocardial infarction (MI) using the CXCR4 antagonist AMD3100 in animal myocardial infarction (MI) models are inconclusive. Chronic AMD3100 administration exacerbated injury and myocardial dysfunction while bolus injection immediately post-MI reduced size of the injury with improvement of systolic function.

Aims:
As MI mobilizes bone marrow stem/progenitor cells and immune cells into peripheral blood which then home to injured myocardium to facilitate and impair, respectively, regeneration and healing we were interested in the role of bone marrow CXCR4 on functional recovery, angiogenesis, and cardiomyogenesis.

Experimental Approach:
To define the role of CXCR4 in the bone marrow we generated chimeras with bone marrow from CXCR4
flox/flox
mice. Wild type mice were transplanted with CXCR4
flox/flox
or CXCR4
flox/flox
UBQ Cre bone marrow cells after a lethal dose of irradiation. CXCR4 deletion was induced 5 weeks after transplant with Tamoxifen. The coronary artery was ligated after another two weeks. Function was evaluated five weeks post MI function by echo and pathology analysis was performed to measure scar size, collagen content, hypertrophy, capillary counts, CSCs counts, angiogenesis and cardiomyogenesis.

Results:
compared to wild type, mice with CXCR4 KO bone marrow had impaired LV systolic function evaluated 5 weeks post-MI by echocardiography. Morphometric analysis of Masson’s Trichrome stained sections confirmed LV exacerbated chamber enlargement (expansion index), larger scar and decreased infarcted wall thickness in mice with bone marrow cells deficient for CXCR4. More detailed analysis revealed c-kit positive CSC numbers were decreased as were proliferating c-kit CSC indicated by Ki67 staining. Decreased proliferation also correlated with reduced numbers of newly formed myocytes (αsarcomeric actin
pos
/BrdU
pos
). Capillary and arteriole counts were also reduced in infarcted hearts of mice with CXCR4 KO bone marrow compared to wild type.

Conclusion:
based on these findings we can conclude that CXCR4 is necessary for bone marrow cell homing to infarcted myocardium to preserve LV function, regenerate lost myocardium, and promote angiogenesis.
Abstract 220: Transcriptional Analysis Of Caveolin And Cavin In The Male And Female Ageing Mouse Heart Following Ischemic Stress
Authors: Kiessling Can J , Reichelt Melissa , Headrick John , Ashton Kevin
Published: July 2021

Cardioprotection against infarction and dysfunction in the myocardium involves G-protein-coupled receptor signalling orchestrated by specialised membrane microdomains termed caveolae. The caveolin protein family consist of three subtypes: caveolin-1, −2 and −3 (Cav1-3) and are responsible for the formation of caveolae and hypothesized to orchestrate cardioprotective signalling. Caveolin-3 deficiency and overexpression has been shown to attenuate and restore cardioprotection, respectively. Recently, a family of four related proteins known as cavins (Cavin1-4) have been implicated as regulators of caveolae formation and function. The roles and expression distribution of the cavin family is currently unknown in cardiac tissue. In this study hearts were isolated from 8, 16, 32 and 48 week male and female mice and subjected to normoxic perfusion (80 min) or ischemic stress (20 min global ischemia, 60 min reperfusion). RT-qPCR was used to assess differential gene expression of caveolin and cavin subtypes across these ages in both sexes.

Decreased post-ischemic pressure development and increased LDH release were observed in 32 and 48 week old relative to 8 week old male hearts hearts, indicative of age-related loss of ischemic tolerance. Females showed greater tolerance to ischemia at 32 and 48 week old hearts when compared to male counterparts. In normoxic male 48 week old hearts, Cav1,-2,-3 and Cavin1 were significantly repressed, whilst post-ischemic male 48 week old hearts demonstrated significant repression of Cav3 and Cavin1 only. Normoxic female hearts showed no significant changes in caveolin and cavin transcript expression over the aging time course. However, post-ischemic female 48 week old hearts showing significant down-regulation of Cav3 only. Taken together, alterations in caveolin and cavin expression may contribute to the age-related loss of ischemic tolerance and G-protein-coupled receptor-mediated protection in aging male and female mice hearts.