bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 262: Depolarization Stimulates Neonatal Cardiomyocyte Proliferation In Vitro
Authors: Williams Corin , Levin Michael , Black Lauren D
Published: July 2021

Cardiac tissue engineering is a promising approach for treating children with congenital heart defects. However, as cardiomyocytes (CMs) undergo a rapid transition from hyperplastic to hypertrophic growth after birth, a major challenge to the development of engineered cardiac tissue is the limited proliferation of CMs. Mature CMs and other terminally differentiated cell types tend to have a highly negative resting membrane potential (Vmem) while stem cells and less mature cells tend to have Vmem closer to zero. Vmem has been shown to play an important role in cell differentiation and proliferation. We hypothesized that depolarization of cardiac cells would stimulate CM proliferation
in vitro
. To test our hypothesis, we isolated neonatal rat cardiac cells and cultured them for 24 hr under standard conditions. Cells were then subjected to depolarization treatment for 72 hr using potassium gluconate or ouabain at various concentrations. Samples were fixed and stained for cardiac α-actin (Fig 1A, red) and phospho-histone H3 (Fig 1A, green) to assess CM mitosis. We found that potassium gluconate had no significant effect while ouabain significantly increased CM mitosis, suggesting Vmem regulation via Na/K-ATPase. CM-specific proliferation was significantly higher with 10nM (p= 0.015) and 100nM (p=0.008) ouabain treatment compared to controls (n=3) (Fig 1B). Cell density was significantly higher with 100μM ouabain versus controls (2656 ± 50 vs. 2026 ± 117 cells/mm
2
), indicating increased cardiac cell proliferation (Fig 1C). Our findings suggest that depolarization promotes CM proliferation and may be a novel approach to encourage growth of engineered cardiac tissue
in vitro
.
Abstract 264: Out-of-Hospital Cardiac Arrest: The Cumulative Effect of Bystander Cardiopulmonary Resuscitation, Automated External Defibrillator and Automated Load Distributing Band Device Use on Return of Spontaneous Circulation
Authors: El Sanadi Nabil , Leduc Todd , Thornton Gabriel , Erdman David , Mansour Jason , Engebretsen Labbe Trine
Published: July 2021

Introduction

: Early bystander C.P.R., A.E.D. use and continuous chest compressions are essential elements of the a.h.a. “chain of survival”. Bystander C.P.R. and A.E.D. use in urban settings are becoming more prevalent. The use of an automated load distributing band (l.d.b.) device for O.H.C.A. management in a large metropolitan area by paramedics was implemented in the winter of 2007; in 9 municipalities, 582 paramedics were trained to use the l.d.b. device as part of a.l.s. measures. A retrospective paramedic “run-sheet” review was performed from 4/2008 - 8/2009 for all nontraumatic adult cardiac arrests. R.O.S.C. on arrival to the emergency departments (e.d.) was the endpoint.

Methods:

86 adult nontraumatic O.H.C.A. rehorts were found. each of the prehospital reports were reviewed. Utstein data elements were collected. bystander C.P.R. with “caller-aided” dispatch, A.E.D. use, paramedic use of standard a.h.a. resuscitation alogarthythms in addition to use of an automated l.d.b. device use (auto pulse® zoll) was recorded. Minitab 15 statistical software was used to evaluate the data. The cumulative effect of sequential “chain-of-survival” components was calculated.

Results:

the mean age was 63 Y.O.; there were 58 males and 28 females. The initial rhythm was: v.fib, pulseless v.tach, P.E.A., or asystole. Chain of survival cumulative synergistic effect on R.O.S.C.

Conclusion:

When bystander C.P.R., in addition to A.E.D. and autopulse were utilized synegistically, R.O.S.C. on e.d. arrival was 71%. When bystander C.P.R. and autopulse were used (without A.E.D. use) only 49% of patients had R.O.S.C. on e.d. arrival. Which is
higher
than previously reported:
Hallstrom
et al. (2006): 26% and
Ong
et al. (2006): 35%. When the l.d.b. device was used with a.l.s., R.O.S.C. was only 20%; which may be due to delayed care; since no efforts were made to resuscitate patients until the paramedics arrived. Our data reaffirms that
early
bystander C.P.R. and A.E.D. use are essential for achieving a high rate of R.O.S.C.
Abstract 267: The Impact of Modulating Afterload on Cardiac Progenitor Cells
Authors: Unno Kazumasa , Bauer Michael , Oikonomopoulos Angelos , Sereti Konstantina , Liao Ronglih
Published: July 2021

Background:

Increased hemodynamic stress induces left ventricular hypertrophy followed by heart failure. Accumulating evidence suggests that the adult mammalian heart possess regeneration ability through the action of resident stem/progenitor cell. However, the effects of hemodynamic fluctuation on cardiac progenitor cell proliferation and cardiac repair remain unknown.

Results:

Proliferation of Cardiac Side Population (CSP) cells and cardiomyocytes was evaluated in experimental models of Ascending Aortic Constriction (AAC)-induced left ventricular hypertrophy (LVH) in mice, over a period of seven weeks. Continuous AAC caused LVH while increasing the number of BrdU
+
CSP cells (one week post-AAC) and the amount of BrdU
+
cardiomyocytes (seven weeks post-AAC). To evaluate the effects of pressure fluctuation on CSP cell and cardiomyocyte proliferation we generated another group (de-AAC) in which the constriction was removed one week post-AAC. Interestingly, restoration of cardiac pressure further increased the proliferative capacity of CSP cells and cardiomyocytes.

Conclusion:

Our data show that restoration of hemodynamic stress confers favorable effects on cardiac repair through increase proliferation of CSP cells and cardiomyocytes. Thus, the hemodynamic state of the heart might be related to CSP cell proliferation and cardiomyogenesis.
Abstract 268: Differential Activation and Subcellular Targeting of Erk1/2 and Akt in Response to β-Adrenergic Receptor-Mediated Transactivation of Epidermal Growth Factor Receptor in Cardiomyocytes
Authors: Grisanti Laurel A , Talarico Jennifer A , Carter Rhonda C , Radcliffe Scott W , Tilley Douglas G
Published: July 2021

β-adrenergic receptors (βAR) are critical regulators of cardiac function whose dysregulation during heart failure are associated with diminished cardiac function, however βAR-mediated EGFR transactivation has been shown to relay cardioprotection via unknown mechanisms. We hypothesized that EGFR transactivation may result in differential activation and subcellular targeting of prosurvival kinases known to be downstream of EGFR, namely ERK1/2 and Akt. Thus, ERK1/2 and Akt phosphorylation and subcellular distribution was assessed in rat neonatal cardiomyocytes (RNCM). Treatment of RNCM with the βAR agonist isoproterenol (Iso) resulted in significant phosphorylation of both ERK1/2 (P-ERK) and Akt (P-Akt), in the cytosolic, plasma membrane and nuclear fractions. EGFR inhibition with AG1478 resulted in complete ablation of Iso-induced P-ERK in all fractions, as did MEK1/2 inhibition with PD184352. Total ERK levels did not change in any fraction under any condition, which along with the PD184352 data suggests Iso-mediated EGFR-dependent effects on ERK1/2 activity at different cellular locations is reliant upon MEK1/2 trafficking. While Akt phosphorylation in response to Iso-mediated EGFR transactivation was not sensitive to EGFR inhibition in the cytosol, the P-Akt response was completely abrogated by AG1478 in the plasma membrane and nuclear fractions. The PI3K inhibitor LY-294002 blocked Iso-induced Akt phosphorylation in all fractions, confirming reliance upon PI3K activity for Iso-mediated Akt activation. Additionally, total Akt levels remained constant over all treatments except at the plasma membrane, where AG 1478 reduced T-Akt, suggesting that Akt recruitment and PI3K activity each contribute to an increase in plasma membrane-associated P-Akt, whereas increased nuclear P-Akt in response to Iso-induced EGFR signaling depends solely on PI3K activity. In all, these results demonstrate differential impact of βAR-mediated EGFR transactivation on the subcellular activation and targeting of cardiomyocyte ERK1/2 and Akt. Further understanding of the downstream consequences of these effects in response to βAR-mediated EGFR transactivation could lead to improved therapies for the treatment of heart failure.
Abstract 278: Targeted Anticancer Drug Sorafenib Impairs Vascular Network Formation by Human Cardiac Microvascular Endothelial Cells
Authors: Konorev Eugene , Sun Zuyue , Taylor Frederick , Malmo Jay
Published: July 2021

Sorafenib is a novel multikinase inhibitor drug that is approved for the treatment of renal cell carcinoma and hepatocellular carcinoma, and is being tested in other common types of cancers. Recent clinical trials revealed that the use of this agent is associated with such cardiovascular complications as hypertension and reduced left ventricular ejection fraction. Sorafenib is known to inhibit Raf kinase, as well as VEGFR2 and PDGF receptor tyrosine kinase activities. We set out to test the hypothesis that sorafenib suppresses cardiac endothelial functions and angiogenesis. We treated human cardiac microvascular endothelial cells (HCMVEC) with VEGF, FGF2, and PDGF-BB in order to examine effects of sorafenib on signaling pathways activated by these pro-angiogenic factors. We determined in these studies that VEGFR2 tyrosine kinase was the most sensitive target of sorafenib in human cardiac endothelial cells. As a result, sorafenib inhibited proliferation and viability of HCMVEC. In order to examine effects of sorafenib on cardiac angiogenesis, we used a co-culture assay of HCMVEC with human cardiac fibroblasts. Sorafenib suppressed the formation of vascular networks in this in vitro assay with an IC50 of approximately 30 nM. In an in vivo Matrigel plug assay, VEGF and FGF2 were admixed into Matrigel before injecting it to mice in order to induce vascular growth within the plug. Control plugs exhibited robust development of vascular networks while sorafenib (daily injections of 30 mg/kg) severely inhibited vascular growth within the plugs. Taken together, this data provide the evidence that endothelial cells present a viable target of sorafenib in the heart.
Abstract 287: Identification of a Novel PKCα Isoform in Heart Transcriptome via RNA Deep Sequencing
Authors: Gao Chen , Lee Jae-Hyung , Xiao Grace (Xinshu) , Wang Yibin
Published: July 2021

The Protein kinase C (PKC) family of Ca2+ and /or lipid-activated AGC kinases are important regulators of multiple cellular responses including cell growth and programmed cell death. Among these, PKCα is the predominant conventional PKC isoform in heart and has been implicated to have a major impact on cardiac function, hypertrophic response and heart failure. Earlier studies have revealed that the conserved AGC-C-terminal tail containing active site cleft is responsible for regulating AGC kinase activity. The phosphorylation events in AGC-C-terminal tail mediate the activity of kinase by regulating kinase maturation, translocation and substrate interaction. In a genome-wide RNA-Seq analysis in mouse heart, we have identified a novel isoform of PKCα-- due to the insertion of a novel exon in its C-terminal tail. This novel exon has a highly conserved sequence from mouse to human, suggesting a conserved role in PKCα regulation. Tissue analysis showed that this PKCα isoform with Novel Exon (PKCαNE) has enriched expression in heart and skeletal muscle, indicating the involvement of a muscle specific alternative splicing event. The insertion of this novel exon lies between the kinase C-Lobe Tether and Active Site Tether. Biochemistry study in cells showed that both PKCα and PKCαNE were activated and translocated from cytosol to membrane and nucleus in response to PMA. However, upon IGF-1 treatment, PKCαNE showed no activation and could not translocate to either membrane or nucleus in contrast to PKCα. To further explore the mechanism for PKCαNE activation, we have determined the phosphorylation events of this novel isoform. Interestingly, the PKCαNE showed a decreased phosphorylation level at the Turn Motif , which could directly impact its activation and maturation.

In summary, we have identified a novel isoform of PKCα that is enriched in heart and skeletal muscle. The PKCαNE isoform has different activation profile and its biological significance in cell signaling in physiology and diseases remains to be further explored.
Abstract 289: Cardiac-Specific Ectonucleoside Triphosphate Diphosphohydrolase 1 Overexpression Affords Protection from Myocardial Infarction and Reperfusion Injury
Authors: Xu Zhaobin , Wheeler Debra G , Mahamud Shouvik D , Dwyer Karen M , Robson Simon C , Cowan Peter J , d'Apice Anthony J , Gumina Richard J
Published: July 2021

Background:
During myocardial stress, extracellular levels of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) increase. These extracellular ATP and ADP levels are modulated via hydrolysis by ectonucleoside triphosphate diphosphohydrolase 1 (ENTDP-1/CD39) to adenosine monophosphate (AMP) subsequently converted by ecto-5'-nucleotidase (CD73) to the anti-thrombotic, cardioprotective nucleoside, adenosine. Previous data demonstrated significantly smaller infarcts in mice globally overexpressing CD39. The current objective was to determine whether tissue specific overexpression of CD39 in the heart would reduce myocardial ischemia/reperfusion injury.

Methods:
Myocardial ischemia/reperfusion (I/R) injury was evaluated in transgenic mice overexpressing human CD39 driven by the α-MHC promoter. I/R injury was induced by ligation of the left anterior descending (LAD) artery for 60 min followed by 24 hours of reperfusion. Myocardial infarct size was determined by staining with triphenyl tetrazolium chloride (TTC) and the area-at-risk was delineated by perfusion with 5% Phthalo Blue.

Results:
Expression of CD39 in the heart tissue was confirmed by Western blot analysis. In response to 60 minutes of ischemia followed by 24 hours of reperfusion, α-MHC CD39-OE animals displayed a marked reduction in infarct size (WT: 31.68%±4.64 vs TG: 6.14%± 2.48, N=5/group, P<0.01), relative to wild-type controls (Figure).

Conclusions:
Overexpression of CD39 in cardiac tissue alone significantly attenuates myocardial ischemic injury.
Abstract 300: The Role of Rheb-mTORC1 Pathway in Cardiac Developmental Growth and Pathological Hypertrophy
Authors: Tamai Takahito , Murakawa Tomokazu , Yamaguchi Osamu , Hikoso Shungo , Komuro Issei , Otsu Kinya
Published: July 2021

Mammalian target of rapamycin (mTOR) is an evolutionary conserved Ser/Thr kinase and plays a key role in cellular growth. Multiprotein complexes called mTOR complex 1 (mTORC1) signaling is essential in cardiac hypertrophy. Many signaling pathways which can regulate mTORC1 activity have been previously reported, however, the regulation of mTORC1 is not fully elucidated. A small GTPase, Rheb (Ras homologue enriched in brain)-GTP activates mTORC1. In this study, to examine the contribution of Rheb in mTORC1 signaling in vivo hearts, we generated floxed Rheb mice to obtain cardiac-specific Rheb-deficient mice. First, to examine the role of Rheb-mTORC1 pathway in developmental cardiac hypertrophy, we generated Rheb-/- mice by crossing Rhebflox/flox mice with alpha MHC-Cre transgenic mice. Rheb-/- were born in Mendelian ratio. Echocardiographic analysis revealed that chamber dimension and contractile function of Rheb-/- were similar compared to those of control mice (Rheb+/+) 5 days after birth. However, all of them died between 8 and 10 days after birth due to cardiac dysfunction and heart failure. Rheb-/- exhibited cardiac dilatation and reduced contractility 8 days after birth. The heart weight to body weight ratio and the cross-sectional area of cardiomyocytes were significantly lower in Rheb-/- 8 days after birth. Next, to examine the role of Rheb-mTORC1 pathway in pathological hypertrophy, we generated conditional Rheb-/- mice by crossing Rhebflox/flox mice with Mer-Cre-Mer transgenic mice. Cardiac pressure overload was induced by means of transverse aortic constriction (TAC), and mice were sacrificed one week after TAC. The heart weight to tibia length ratio and the cross-sectional area of cardiomyocytes were significantly increased in both TAC-operated control and conditional Rheb-/- group compared to that of corresponding sham-operated group. Taken together, Rheb is not essential in pathological hypertrophy, but is essentilal in cardiac developmental hypertrophy in the post-neonatal period.
Abstract 304: Global Transcriptional Effects of Vitamin A Deficiency on the Postnatal Heart
Authors: Galindo Cristi L , Tran Truc-Linh , Peng Xuyang , Sawyer Douglas B , Asson-Batres Mary
Published: July 2021

Vitamin A (VA) is the chemical precursor of retinoic acid (RA), which is critical for embryonic development and for growth, immunity, metabolism, and cell differentiation in postnatal regenerating systems such as skin, sensory organs, and stem cell niches in the brain. VA is also essential for embryonic heart development, and we hypothesized that Vitamin A might exert an effect on the postnatal heart similar to what is observed for other tissues. Here, we report the global transcriptional profiles of wild-type (WT) mice fed a VA sufficient diet (VAS) compared with retinyl acyl transferase (LRAT) knock-out mice fed either a VAS or VA deficient (VAD) diet. Knockout of the LRAT gene alone was sufficient to induce differential expression of 576 genes relative to WT. Feeding LRAT mutant mice a VAD diet resulted in a change in the relative expression levels of 257 genes relative to LRAT mutant mice fed a VAS diet. As expected, we observed transcriptional alterations related to Vitamin A metabolism, including an increase in the gene encoding cellular retinoid binding protein 7 and down-regulation of the retinol metabolic enzymes Cy1a2 and Cyp2a4. Importantly, several cardiac genes not previously known to require VA were perturbed, including the gene encoding B-type natriuretic peptide, which was down-regulated in mutant mice irrespective of diet, and A-type natriuretic peptide, which was decreased only in mice fed the VAD diet. There was also a striking effect of VAD on genes important for immune responses, which could have an impact on the wound healing process subsequent to injury of the heart. This is consistent with recent evidence that showed that Vitamin A deficiency influences post-infarct ventricular remodeling in rats. In summary, this is the first microarray study of Vitamin A deficiency in the postnatal heart, which suggests mechanisms by which Vitamin A depletion may alter myocardial maintenance and repair after injury.
Abstract 306: Proinflammatory Prostaglandin E Impairs Adrenergic Contractility in Animal Hearts
Authors: Xiang Yang K
Published: July 2021

Introduction:

Inflammation is a major risk factor for heart failure; inflammatory prostaglandin E is elevated in myocardium, and has been associated with development of heart failure via cardiac LV remodeling, but the mechanisms remain unclear.

Hypothesis:

We hypothesize that proinflammatory prostaglandin signaling might affect Left ventricular contractility by directly impairing β-adrenergic (βAR) signaling.

Methods and Results:

Pretreatment with Prostaglandin E impairs intracellular PKA phosphorylation of substrates in myocardium and cardiac contractile responses under β-adrenergic stimulation. Both prostaglandin E and β-adrenergic agonist isoproterenol induce intracellular cAMP activities in myocytes. However, the cAMP signal under prostaglandin stimulation is confined along the plasma membrane whereas the cAMP signal under isoproterenol stimulation is distributed throughout the cells, including the intracellular sarcoplasmic reticulum (SR), a calcium storage compartment critical for myocyte calcium signaling and contractile response. Interestingly, the prostaglandin E-induced cAMP signal is sufficient for PKA activation, leading phosphorylation and activation of phosphodiesterase 4 on the plasma membrane, a group of enzymes associated with membrane βARs for cAMP degradation. Moreover, pretreatment of prostaglandin E prevents dissociation of phosphodiesterase 4 from βAR induced by isoproterenol, a step necessary for cAMP diffusion from the plasma membrane to the intracellular SR. Together, prostaglandin E induces activation of phosphodiesterase 4 in βAR complex and prevents the cAMP diffusion from the plasma membrane to the SR under isoproterenol stimulation, and inhibits calcium signaling and contractile response in myocytes and animal hearts.

Conclusions:

This study provides a novel mechanism underlying the phosphodiesterase-mediated signaling crosstalk between two neurohormonal stimulation in myocardium under chronic conditions, and suggests that proinflammatory prostaglandin signaling may impair β-adrenergic contractile in the development of heart failure.
Abstract 308: Chronic Aspirin Treatment Possibly Poses a Risk to Ischemic Heart via Impairment of Cardiac Proteasome Functions
Authors: Tan Chunjiang , Chen Wenlie , Wu Yanbing , Lin Jiumao , Lin Ruhui , Tan Xuerui , Chen Songming
Published: July 2021

Background:
Impaired cardiac proteasome (CP) has been reported in ischemia and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, it’s unclear whether it affects CP functions.

Objective:
We investigated the influence of aspirin on CP in the rat model of myocardial infarction (MI).

Methods and Results:
MI, sham or normal male SD rats were injected intraperitoneally with high (300mg/kg), low (5mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated cell model of primary human ventricular myocytes incubated with different concentrations of aspirin. Myocardial hypertrophy, cardiac function, cell viability and the functions of 26S, 20S and 19S, including the β1, β2 and β5 subunits of 20S were determined. The activity of 26S, 20S and 19S declined by about 30%, and β5, β2 and β1 by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats (
P
<0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, and β5 further decreased by 30% and 12%, and β1 by 40% and 30%, respectively, and their lost activity was correlated with compromised cardiac function in the MI rats. The dose-related and selective inhibition of β5 and β1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. However, the CP in the normal settings, or 19S and β2 in all the groups of animals or cultured cells were less affected by aspirin treatment. The impaired CP, dose-dependently enhanced by aspirin, led to the elevation of oxidative and ubiquitinated proteins in the MI rat hearts or in the hypoxia/reoxygenated cells. These aberrant proteins in turn constituted the ultimate causal factor for the cardiac dysfunction and the loss of cell viability. In vitro experiments confirmed that the purified CP from the MI rats or hypoxic cells, but was more susceptible to aspirin treatment, whereas the complexes in the normal settings appeared less affected by aspirin.

Conclusions:
Chronic aspirin treatment,
via
the dose-dependent and selective inhibition of CP, enhanced the ischemic CP dysfunction, which constitutes a potential risk factor for the ischemic heart.
Abstract 316: Effect of Different Substrates on Functional Performance and Kinase Activation During Reperfusion After Ischemia in Hearts from Obese Insulin Resistant Rats
Authors: Lochner Amanda , Fan Wen-Jun , Huisamen Barbara
Published: July 2021

Insulin resistance decreases glucose uptake, alters lipid metabolism and impairs PKB-dependent signaling in its target tissues. In view of the myocardial dependence on glycolysis during energy deficiency, insulin resistance may exacerbate the harmful effects of ischaemia. We hypothesize that obesity-induced insulin resistance affects functional recovery and activation of PKB, JNK, ERK and PTEN during early reperfusion.

Methods:
Insulin resistance was induced by feeding rats a high calorie diet for 16 weeks. Hearts from diet rats (D) and age-matched controls (C) were subjected to (i) 15 min global ischaemia and different reperfusion times for Western blot and measurement of functional recovery, or (ii) 35 min regional ischaemia and 2 hours reperfusion for infarct size determination (IS) using tetrazolium. Substrates were glucose (G) (10mM) or glucose (10mM) plus fatty acid (FA) (1.2mM palmitate /3%BSA) (G+FA). N=4-8/group.

Results:
Regardless of the substrate, functional recovery during reperfusion of hearts from D was higher than those of C (% recovery of total work: D+G/C+G, 69.1±3.4/50.7±4.5, D+G+FA/C+G+FA: 105.4±3.6/92.7±3.9), associated with a reduction in IS (D+G/C+G 29.7±2.8/43.5±2.5, p<0.05; D+G+FA/C+G+FA 26.7±3.2/36.6±4, p<0.05). When perfused with glucose hearts from D showed increased activation of PKB and JNK at different times during reperfusion (D+G/C+G: 10 min PKBs473, 1.41±0.11/1.00±0.06; JNKp54, 1.80±0.15/1.00±0.13; JNKp46, 1.59±0.11/1.00±0.08). Addition of FA caused further activation of the kinases (e.g. D+G+FA/D+G: 5 min ERKp44, 1.54±0.11/1.00±0.11, ERKp42, 1.46±0.13/1.00±0.07; 10 min ERKp44, 1.83±0.19/1.00±0.05, ERKp42, 1.27±0.10/1.00±0.05; 30 min PKBs473, 2.91±0.35/1.00±0.09) and inhibition of PTEN (1.79±0.11/1.00±0.10).

Conclusion:
Contrary to expectations, hearts from D rats showed an increased tolerance to ischaemia/reperfusion (I/R) injury which was further improved by fatty acids in the perfusate. The pattern of PKB, JNK, ERK and PTEN phosphorylation during reperfusion is substrate dependent and significantly higher in hearts from D rats. With glucose as substrate activation of PKB and JNK during reperfusion was higher in hearts from D rats while FA further improved activation of ERK and PKB.
Abstract 318: Heterogeneous Remodeling in Diabetic Hearts Leads to Sudden Cardiac Death
Authors: Panguluri Siva K , Tur Jared , Tipparaju Srinivas
Published: July 2021

Diabetes increases propensity to sudden cardiac death. Although ion channels were implicated, the precise mechanisms leading to arrhythmias remain unclear. We hypothesized that the diabetic heart undergoes heterogeneous remodeling leading to arrhythmogenic substrate and sudden cardiac death. For this, we investigated the expression of cardiac specific microRNAs, key transcriptional factors and their target gene in right (RV) and left ventricle (LV). TaqMan and SYBR green qRT-PCR was used to estimated the transcriptional data and Western blots/ELISA for protein profiles. Our data clearly suggests that miR-1-1a (1.6±0.021; p≤0.00067) and miR-301 (1.36±0.02; p≤0.0201) were up-regulated in epicardium of left ventricle (LV-epi), whereas miR-1-1a (2.9±0.59; p≤0.027), miR-133a (3.7±0.19; p ≤ 0.041) and miR-301 (3.6±0.26; p-value ≤ 0.037) were up-regulated in RV of diabetic (db/db) mice (n=3) hearts compared with wild type (n=3). Out of 30 genes tested we confirmed down-regulation of 14 genes (with p≤0.05) including transcriptional factors NFkB2, GATA6, Irx5, Ezh-2, Six-1, CtBP, SiRT1; ion channels Kv4.2, Scn1b and Scn5a and its protein chaperon Kchip-2, whereas Mef2c was up-regulated significantly in LV-epi in diabetic mice. Similarly, 11 genes including Kv4.2, Mef2c, GATA4, GATA6, Pitx2c, Scn1b, Kchip-2, SiRT1, Nampt and MMP-9 were down-regulated (p≤0.05), whereas 9 genes such as Irx5, Kv2.1, Kv4.3, NFkB1, NFkB2, MHC7, CtBP, NNt, and G6PD were up-regulated significantly in RV of diabetic mice. The Ingenuity Pathway Analysis of the differentially expressed genes in heart revealed hypertrophy, arrhythmias, proliferation, and failure as top toxicology networks in LV-epi, whereas cardiac hypertrophy, hepatic stellate cell activation, oxidative stress, PPAR mediated gene activation, fibrosis and necrosis are among top toxicology networks in RV. The role of miR’s along with transcriptional factors down-regulating Kv4.2 in diabetic LV may be consistent with the idea that repolarization reserve is decreased in diabetic hearts. Moreover, regional differences in potassium current may play vital role in regulating function. These data suggest that the ion channel remodeling in left and right ventricles are modulated via different mechanisms.
Abstract 33: Endoplasmic Reticulum Stress and Vascular Dysfunction in Hypertension
Authors: Kassan Modar , Galan Maria , Partyka Megan , Henrion Daniel , Trebak Mohamed , Matrougui Khalid
Published: July 2021

Objective:
Cardiac damage and vascular dysfunction are major causes of morbidity and mortality in hypertension. In this study, we explored the beneficial therapeutic effect of endoplasmic reticulum (ER) stress inhibition on cardiac damage and vascular dysfunction in hypertension.

Methods & results:
Mice were infused with angiotensin II (Ang-II, 400 ng/kg/min) with or without ER stress inhibitors (Tudca and PBA) for two weeks. Mice infused with Ang-II displayed an increase in blood pressure, cardiac hypertrophy and fibrosis associated with enhanced collagen-I content, TGFβ1 activity, and ER stress markers, which were blunted after ER stress inhibition. Hypertension induced ER stress in aorta and mesenteric resistance arteries (MRA), enhanced TGFβ1 activity in aorta but not in MRA, and reduced eNOS phosphorylation and endothelium-dependent relaxation (EDR) in aorta and MRA. The inhibition of ER stress significantly reduced TGFβ1 activity, enhanced eNOS phosphorylation and improved EDR. The inhibition of TGFβ1 pathway improved EDR in aorta but not in MRA, while the reduction in ROS levels ameliorated EDR in MRA only. Infusion of tunicamycin in control mice induced ER stress in aorta and MRA, and reduced EDR by a TGFβ1-dependent mechanism in aorta and ROS-dependent mechanism in MRA.

Conclusion:
ER stress inhibition reduces cardiac damage and improves vascular function in hypertension. Therefore ER stress could be a potential target for cardiovascular diseases.
Abstract 346: Lipophilic Statins Attenuate Human Atrial Fibroblast Viability In Vitro
Authors: Sran Kiranjit K , Li Yun , Ghavami Saeid , Ngo Melanie , Arora Rakesh C , Kirshenbaum Lorrie , Freed Darren
Published: July 2021

Cardiovascular diseases (CVD) leading to heart failure are associated with myocardial cell loss and cardiac fibrosis. Hydroxymethylglutaryl-Coenzyme-A Reductase (HMGR) inhibitors ("statins") are widely used to limit cardiovascular events in patients with hypercholesterolemia and CVD by altering their lipid profile. HMGR inhibition reduces cholesterol precursor L-mevalonate production, whose depletion induces autophagy, apoptosis, and endoplasmic reticulum stress in various cell types. However it is unclear if this is a class effect or a phenomenon specific to various compounds. We examined the in vitro effect of HMGR inhibition on human atrial fibroblast (hATF) viability with particular reference to hydrophilic vs lipophilic compounds.

Hypothesis- Lipophilic statins induce cell death in primary hATF via mevalonate depletion; whereas hydrophilic statins do not have any effect on hATF viability.

IRB approval was obtained for collection of hATF from consenting patients undergoing open heart surgery. Cells were treated with atorvastatin, simvastatin or pravastatin (0.1, 1.0 or 10 λM) for 24, 48, 72 or 96 hours. Expression of proteins involved in the regulation of apoptosis and autophagy was assessed using immunoblotting. Cell viability was assessed using MTT assay.

Treatment of hATF with 0.1 - 10 λM atorvastatin or simvastatin (lipophilic statins) resulted in progressively reduced cell viability in time and dose-dependent manner. Viability could be rescued by coincubation with mevalonate. Expression of key apoptotic cascade proteins -Bcl2, Bax and cleaved Caspase3 showed a clear induction of apoptosis. Also, there was an increase in Atg5-12 expression at 24h indicating induction of early autophagic response. Pravastatin (hydrophilic statin) did not affect cell viability or autophagy and apoptosis.

We conclude that statin-induced cell death is mediated by mevalonate depletion, which activates intrinsic apoptotic pathways in hATF. Lipophilic statins impair the viability of hATFs in vitro, whereas hydrophilic statins have no effect on cell growth and cell viability of hATFs. This may represent an additional pleiotropic effect of statins, and may represent a novel therapeutic option for the prevention and treatment of cardiac fibrosis.
Abstract 352: Myocardial Neuregulin-1/ErbB Signaling Is Impaired in the Setting of Post--Myocardial Infarction Heart Failure Complicated by Type 1 Diabetes Mellitus
Authors: Odiete Oghenerukevwe , Dennis Kathleen E , Sawyer Douglas B , Hill Michael F
Published: July 2021

Background:
Type 1 diabetes mellitus (DM) patients surviving myocardial infarction (MI) are at heightened risk for the subsequent development of heart failure (HF). Despite the worse outcomes, investigations into the pathophysiological mechanisms that contribute to the increased frequency of HF after MI in the type 1 DM heart remain scarce. Neuregulin-1 (NRG-1), along with the ErbB family of receptor tyrosine kinases through which NRG-1 ligands signal, have been shown to be intimately involved in mediating cardiac recovery after MI. However, the impact of type 1 DM on this signaling system post-MI remains to be elucidated. Therefore, in the present study, we examined myocardial NRG-1/ErbB signaling during post-MI HF in the presence of type 1 DM.

Methods:
Type 1 DM was induced in male Sprague-Dawley rats via a single intraperitoneal injection of streptozotocin (STZ) (65 mg/kg). Two weeks after induction of type 1 DM, MI was produced in DM and non-DM rats by ligation of the left anterior descending (LAD) coronary artery. Residual left ventricular (LV) function was assessed by echocardiography at 4 weeks post-MI. Following echocardiographic assessment, NRG-1, ErbB2, and ErbB4 protein expression was assessed in the remote, surviving LV myocardium of DM and non-DM rats using Western blot techniques.

Results:
LV Fractional Shortening (FS) and LV Ejection Fraction (EF) were significantly lower in the DM + MI group compared to the MI group ([LVFS: DM + MI, 17.9 ± 0.7 (n=6) vs. MI, 25.2 ± 2.2 (n=6),
p
<0.05; LVEF: DM + MI, 35.5 ± 1.4 (n=6) vs. MI, 47.5 ± 3.5 (n=6),
p
<0.05]), indicating an increased functional severity of HF in the diabetic post-MI group. The weight of myocardial scar caused by the infarction was not significantly different between the MI groups ([DM + MI, 0.19 ± 0.02 g (n=4) vs. MI, 0.20 ± 0.03 g (n=4),
p
=0.70]). ErbB2, ErbB4, and NRG-1 protein expression levels were all significantly lower in the DM + MI group compared to the MI group.

Conclusions:
These findings demonstrate that type 1 DM impairs myocardial NRG-1/ErbB signaling in response to MI, which may contribute to the accelerated progression of subsequent HF. Augmentation of NRG-1 or its downstream signaling pathways may represent a novel therapeutic strategy for ameliorating post-MI HF in the setting of type 1 DM.
Abstract 369: Isolation and Characterization of Developmentally and Functionally Discrete Subsets of Cardiac Fibroblasts
Authors: Ardehali Reza , Ali Shah , Weissman Irving
Published: July 2021

Although cardiac fibroblasts (CF) are the most prominent cell types in the heart, little is known about their origin and development. Fibroblasts play a key role in regulating the normal myocardial function, as well as the adverse remodeling that occurs with injury. Fundamental to understanding cardiac development is the ability to determine when and where CFs are generated, their ancestry, and how they move to reside in their final position. We used novel transgenic mouse models to lineage trace the developmental origin of CFs and their contribution to fibrosis in response to injury. Here, we show that a subset of cardiac fibroblasts is derived from Mesp1-expressing cells (multipotent cardiac progenitor cells that contribute to precursors of both heart fields). Using Mesp1-Cre;mT/mG mice, in which cells derived from Mesp1-expressing cells are indelibly marked by the GFP reporter protein, we demonstrate that approximately 65% of CFs share an embryonic origin with cardiomyocytes, vascular smooth muscle, and endothelial cells. In addition, experimental myocardial fibrosis did change this proportion. In an attempt to identify the source of the fibroblasts that are non-MesP1-derived, we evaluated contribution from: (i) the bone marrow stromal cells and hematopoietic stem cells, (ii) endothelial-to-mesenchymal transition, (iii) circulating cells, and (iv) epicardial-derived cells. Transplantation of GFP-bone marrow into irradiated wildtype mice resulted in an insignificant contribution of stromal-derived fibroblasts in the heart in response to injury. Using Tie2-Cre;mT/mG mice, we did not observe cardiac fibroblasts originating from endothelial cells in injured hearts. Finally, using a parabiotic pair of GFP and wildtype mice where blood chimerism is established, no evidence for homing of circulating fibroblasts to the heart upon injury was noted. However, we provide unequivocal evidence that epicardial-derived cells migrate to myocardium as fibroblasts to contribute to fibrosis. In summary, using lineage-tracing systems, we provide evidence for two sources of fibroblasts in the heart, one that shares an embryonic origin with the cardiovascular lineages and the other from a non-cardiac origin, which is primarily derived from the epicardium.
Abstract 39: Organic Nitrates Improve Calcium Handling and Myofilament Sensitivity Impaired by Nitroso-Redox Disequilibrium
Authors: Dulce Raul A , Balkan Wayne E , Hare Joshua M
Published: July 2021

Oxidative stress is a key factor in the dysfunctional calcium (Ca
2+
) handling and contractile performance in heart disease. Organic nitrates are used for the treatment of cardiovascular afflictions, however endogenous nitric oxide (NO) has a broad spectrum of actions and the mechanisms by which it regulates Ca
2+
cycling and contractility is poorly understood. Neuronal NO synthase (nNOS) deficiency induces a nitroso-redox disequilibrium characterized by oxidative stress and altered contractility and calcium handling. We hypothesize that treatment of nNOS
−/−
mice with organic nitrates such as nitroglycerin (TNG) or isosorbide dinitrate (ISDN) reduces cytosolic calcium levels and restores myofilament responsiveness to calcium with no changes in contractility.

Cardiomyocytes (CMs) were isolated from nNOS
−/−
(N=7) and wild type (WT) mice (N=3). Cells were loaded with fura-2 and then electrically evoked intracellular Ca
2+
and sarcomere length were simultaneously measured in an IonOptix system. It has been shown that L-type Ca
2+
current is exacerbated in nNOS
−/−
cardiomyocytes. In consequence, at 1 Hz pacing rate, Ca
2+
peak and transient amplitude were increased in nNOS
−/−
(Peak Ca
2+
: 571 ± 38 nM) compared to WT (401 ± 32 nM;
p
= 0.012) cardiomyocytes, which was reduced toward normal by 10 nmol/L TNG (453 ± 55 nM;
p
= 0.11) as well as 10 nmol/L ISDN (394 ± 65;
p
= 0.035). Phospholamban phosphorylation has been shown to be reduced in nNOS
−/−
CMs, possibly due to the enhanced activity of protein phosphatases induced by oxidative stress. Thus, Ca
2+
decay and sarcomere relaxation were slower in nNOS
−/−
compared to WT CMs (
p
< 0.0001). Treatment with TNG (
p
< 0.0001 vs. NOS1
−/−
) or ISDN (
p
< 0.0001 vs. NOS1
−/−
) accelerated Ca
2+
decay and relaxation. Ca
2+
sensitivity was impaired in nNOS
−/−
(
p
< 0.021 vs. WT). Although organic nitrates either reduce or do not affect myofilament sensitivity in normal myocytes, TNG and ISDN increased their responsiveness to Ca
2+
in nNOS
−/−
.

In conclusion, restoration of NO availability and subsequent attenuation of the nitroso-redox imbalance, improved excitation-contraction coupling in nNOS
−/−
CMs, decreased intracellular Ca
2+
which is counteracted by the improved myofilament sensitivity resulting in no net change in contractility.
Abstract 42: In Vivo Differentiation of Epigenetically Reprogrammed Human Endothelial Progenitor Cells into Cardiomyocytes Enhances Functional and Anatomical Postinfarct Myocardial Repair
Authors: Thal Melissa A , Krishnamurthy Prasanna , Mackie Alexander R , Hoxha Eneda , Lambers Erin , Verma Suresh , Ramirez Veronica , Kishore Raj
Published: July 2021

Currently, bone marrow derived endothelial progenitor cells (human CD34+ cells, EPC) are being used clinically to improve vascularization in patients with ischemic heart disease. While it is generally accepted that CD34+ cells predominantly work through a paracrine mechanism, there exists no convincing evidence that these cells trans-differentiate into functional cardiomyocytes (CMC). Since ischemic heart disease leads to substantial loss of CMC, improving cardiomyogenic plasticity of an existing autologous cell therapy is of obvious import. EPC and CMC both differentiate from a common mesodermal progenitor however; during EC-specific lineage differentiation, CMC specific genes are epigenetically silenced. We hypothesized that reprogramming of CD34+ cells using small molecules targeting key epigenetic repressive marks may recapitulate their cardiomyogenic potential.

Human CD34+ EPCs were treated with inhibitors of histone deacetylases (valproic acid) for 24 hours followed by an additional 24 hours with the DNA methyltransferase inhibitor (5-Azacytidine). This forty-eight hour treatment led to the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific gene expression was maintained. Intra-myocardial transplantation of a sub-therapeutic dose of reprogrammed CD34+ cells in an acute myocardial infarction mouse model showed significant improvement in LV function compared to the same number of control CD34+ cells that are therapeutically equivalent to no treatment at all. This was histologically supported by de novo CMC differentiation. In addition to increased cardiomyogenic plasticity, drug treatment also enhanced the inherent therapeutic capacity of the CD34+ cells as shown by reduced fibrosis, increased capillary density, increased proliferation, increased cell survival and increased secretion of angiogenic factors.

Taken together, our results suggest that epigenetically reprogrammed CD34+ cells are “super-CD34+ cells” that have an enhanced paracrine effect, display a more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.
Abstract 47: GATA Factors Caught at a Crossroad of Gene Duplication with Functional Divergence
Authors: van Berlo Jop H , Molkentin Jeffery D
Published: July 2021

Background
Six individual members comprise the GATA family of Zn finger-containing transcription factors that play major roles in the hematopoietic system and many mesoderm and endoderm derived tissues. The adult heart expresses both GATA4 and GATA6. Here, we examined the overlapping and diverging functional roles of GATA4 and GATA6 in the adult heart, both at baseline and under stress.

Results
Pressure overload by transverse aortic constriction (TAC) caused a blunted hypertrophic response when GATA4 was deleted from the adult heart, with severe heart failure ensuing after 4 weeks. Similarly, deletion of GATA6 from the mouse heart showed a blunted hypertrophic response and heart failure. Next, we deleted 1 allele of GATA4 and 1 allele of GATA6 from the adult heart, also resulting in blunted hypertrophy and cardiac dysfunction. Deletion of all four alleles of GATA4 and 6 resulted in spontaneous heart failure and death by 3 months of age.

These results suggested functional overlap or synergistic activation. To address this concept more directly we deleted GATA6 from the adult heart and overexpressed either GATA4 or GATA6 in a cardiac-specific manner. As expected, we were able to completely revert the phenotype to wild type when GATA6 was overexpressed in mice that had GATA6 genetically deleted. Surprisingly, overexpression of GATA4 was unable to rescue the absence of GATA6 and actually worsened cardiac function in response to pressure overload. Possible explanations for this functional divergence were suggested by an observed rarefaction in capillaries of the heart in absence of GATA4, but enhanced angiogenesis in absence of GATA6. Moreover, when we induced cardiac hypertrophy through MAPK activation, we observed a critical necessity for GATA4, while GATA6 was dispensable.

Conclusion
Although GATA4 and 6 may be functionally complementary for cardiac gene expression and hypertrophy, they evolved some specific roles in the heart, such as angiogenesis and stress activation. We are currently unraveling how GATA4 and 6 may differentially regulate genes.
Abstract 73: Aberrant Activation of γ2-AMPK Increases Cardiac Growth Through Cellular Hypertrophy and Hyperplasia
Authors: Kim Maengjo , Hunter Roger , Sakamoto Kei , Kolwicz Stephen , Menendez Lorena , Gong Guohua , Wang Wang , Seidman Christine , Seidman Jonathan , Tian Rong
Published: July 2021

AMP-activated protein kinase (AMPK) is an energy sensor and a key regulator of cell metabolism, hence a promising drug target. Point mutations in the regulatory γ2-subunit (encoded by
PRKAG2
gene) have been shown to cause a unique form of cardiomyopathy in humans characterized by cardiac growth, arrhythmias and glycogen storage. In previous studies, we demonstrated that the mutation of prkag2 (N488I) caused aberrant activation of AMPK leading to glycogen storage. However, elimination of glycogen storage by inhibiting glycogen synthase activity failed to normalize heart weight (HW) of the mutant mice. Here, we aimed to determine whether cardiac growth in PRKAG2 ardiomyopathy was dueto cellular hypertrophy or hyperplasia. We used transgenic mice expressing a mutant
PRKAG2
(N488I) in the heart (TGγ2
N488I
) that faithfully recapitulated
PRKAG2
cardiomyopathy. We determined HW and cardiac myocyte size in adult (2 months) and postnatal (2 weeks) hearts in WT and TGγ2
N488I
. At 2 months, TGγ2
N488I
hearts show a 2.4-fold increase in HW/BW (body weight) (10.3 ± 1.44 vs. 4.3± 0.17 mg/g) as well as cross-sectional cell surface area compared WT hearts (325 ± 13 vs. 155 ± 5.4 μm
2
,p<0.01), suggesting cellular hypertrophy in adult TGγ2
N488I
heart. Furthermore, we observed increased mTOR activity evidenced by enhanced phosphorylation of mTOR (Ser2448) as well as its downstream targets S6 and 4E-BP. The HW of TGγ2
N488I
was partially inhibited by treatment with rapamycin, an inhibitor of mTOR. Interestingly, the length and width of isolated cardiomyocytes from 2 weeks old mice were not different between the WT and TGγ2
N488I
heart in spite of a 50% increase of HW of TGγ2
N488I
mice. We observed a 2- fold increase in the expression of a proliferation marker, proliferating cell nuclear antigen (PCNA) during postnatal cardiac growth. Expression of Cyclin genes including cyclin D1, D2 and E1 was greatly increased in TGγ2
N488I
hearts (2.4 - 4 fold, p<0.01 vs. WT). Taken together, these data indicate that aberrant γ2-AMPK activation stimulates cardiac growth through increased cell number during postnatal growth period and increased cell size at adulthood. These results suggest a novel role of γ2-AMPK in the growth of cardiac myocytes.