bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 203: Protein Synthesis Inhibitors 4E-BPs Regulate Cardiac Function
Authors: Zhang Denghong , Contu Riccardo , Latronico Michael , Peterson Kirk , Chen Ju , Condorelli Gianluigi
Published: July 2021

Rationale:
mTOR regulates cardiac functionality and hypertrophy. We have shown that cardiac-specific deletion of mTOR induces fatal dilated cardiomyopathy. This model of heart failure is characterized biochemically by accumulation of the mTOR substrate 4E-BP1, which inhibits protein synthesis through the regulation of translation initiation. Deletion of the 4E-BP1 gene, Eif4ebp1, in mTOR-KO mice significantly, albeit partially, improved cardiac function and survival.

Objective:
To investigate the role of the 4E-BP family in the heart by determining whether absence of its members improves cardiac function in different models of heart failure.

Methods and Results:
In the absence of Eif4ebp1, cardiac expression of the 4E-BP2 homolog increases strikingly. Deletion of Eif4ebp2 along with Eif4ebp1 (4E-BP1/4E-BP2-dKO mice) abrogated the negative effects of the loss of mTOR on cardiac function, and led to a further significant improvement in survival rate. Moreover, when 4E-BP1/4E-BP2-dKO mice where subjected to pressure-overload stress, cardiac function and survival were significantly improved compared with similarly treated control mice. The hypertrophic response to pressure overload in these mice was not affected by the absence of 4E-BPs.

Conclusions:
Our data indicate that 4E-BPs are responsible for an important part of mTOR effects on cardiac function, further strengthening the concept that the regulation of mRNA translation profoundly affects cardiac inotropism under stress.
Abstract 204: Inhibition of Histone Deacetylase Activity Represses Matrix Metalloproteinase-9 Induction Through Regulation of Macrophage Polarization
Authors: Kimbrough Denise M , Mani Santhosh K , Kern Christine B , Spinale Francis G , Mukherjee Rupak , Menick Donald R
Published: July 2021

Post myocardial infarction (MI) the extracellular matrix (ECM) undergoes massive remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Integral players in this remodeling are matrix metalloproteinases (MMP). Post-MI macrophages are a significant source of MMP production and have been shown to have temporal expression of the two phenotypes (M1 and M2). Differential expressions of macrophage phenotypes correlate with the different phases of ECM remodeling, ECM composition and MMP expression. Increased expression of MMP-9 is associated with deleterious effects to the ECM and left ventricular (LV) dilation following MI. MMPs major mechanism of expression regulation is transcription. Histone deacetylases (HDAC) are a class of enzymes that can greatly affect transcriptional regulation of genes during pathological conditions. Therefore, we hypothesize that HDAC inhibition controls the post-MI expression of MMP-9 by mediating macrophage polarization to improve LV function. The post-MI change in LV end-diastolic volume is significantly lower with treatment of the class I and IIb HDAC inhibitor trichostatin A (TSA) and ejection fraction is improved in mice. Immunohistochemical analysis and zymography revealed that MMP-9 expression at 7 days post-MI is attributable to infiltrating macrophages. Immunoblotting shows that TSA inhibits control levels and lipopolysacharide (LPS) stimulated upregulation of MMP-9 in cultured macrophages, while the class I specific inhibitor, PD106, only inhibits control. The HDAC 6 inhibitor, Tubastatin A (Tub A), partially inhibits LPS stimulated upregulation of MMP-9, but surprisingly appears to potentiate control levels of MMP-9. Immunofluorescence revealed that direct HDAC inhibition with PD106, Tub A, and TSA leads to M1 to M2 macrophage polarization and maintenance of anti-inflammatory, M2, phenotype even with LPS stimulation in culture. From these data we conclude that macrophage secretion of MMPs is an essential component in the promotion of adverse ECM remodeling post-MI. HDAC inhibition can attenuate this remodeling possibly through macrophage polarization, but HDAC inhibition mediated M2 polarization may not be sufficient to suppress MMP-9 expression.
Abstract 205: Differing Cardiac Phenotypes Between PP1α and PP1β Heart-Specific Gene-Deleted Mice
Authors: Messier Mannix , Liu Ruijie , Molkentin Jeffery
Published: July 2021

Background:
Protein phosphatase 1 is the major protein serine/threonine phosphatase in nearly all mammalian tissues, where it consists of three isoforms PP1α, PP1β, and PP1γ. However, the redundant or specific roles of each isoform in the heart is not known

Methods:
Each PP1 isoform was conditionally deleted in the mouse heart using a Cre-loxP approach. LoxP sites were introduced into intron 1 and 3 of each PP1α and PP1β. Both loxP-targeted lines were bred with mice expressing β-myosin heavy chain promoter driven Cre to achieve isoform specific gene deletion in the heart. Echocardiography was performed in these mice at different ages. We also investigated protein phosphorylation status of selected PP1 targets that underlie cardiac contraction and calcium handling from the hearts of these deleted mice.

Results:
Heart-specific deletion of PP1α caused a reduction of fractional shortening and worsening of cardiac function. Two weeks after transaortic constriction (TAC), PP1α deleted mice had greater increases in heart-weight to body-weight ratio compared with control mice, suggesting that PP1α was important for proper cardiac compensation. Interestingly, however, combined deletion of both PP1α and PP1β rescued the cardiac performance defects observed in PP1α deleted mice. Mechanistically, we found that deletion of PP1αβ led to increased phospholamban serine 16 and threonine 17 phosphorylation compared to that of PP1α. In conclusion, we showed that PP1 isoforms play distinct roles in the heart in regulating contractility and compensation after pressure overload stimulation.
Abstract 206: A New MicroRNA Family Regulating Cardiac Autophagy and Hypertrophy
Authors: Thum Thomas , Gupta Shashi K , Ucar Ahmet , Fiedler Jan , DeWindt Leon , Engelhardt Stefan , Bauersachs Johann , Chowdhury Kamal
Published: July 2021

Pathologic growth of cardiomyocytes and derailed autophagy are major determinants for the development of heart failure, one of the leading medical causes of mortality worldwide. Here, we show the microRNA (miRNA)-212/132 family to regulate hypertrophy and autophagy in cardiomyocytes. Hypertrophic stimuli lead to the upregulation of miR-212 and miR-132 expression in cardiomyocytes, which are both necessary and sufficient to drive the hypertrophic growth of cardiomyocytes. MiR-212/132 null mice are protected from pressure-overload induced heart failure, whereas cardiomyocyte-specific overexpression of the miR-212/132 family leads to pathological cardiac hypertrophy, heart failure and lethality in mice. Mechanistically, both miR-212 and miR-132 directly target the anti-hypertrophic and pro-autophagic FoxO3 transcription factor and overexpression of these miRNAs leads to hyperactivation of pro-hypertrophic calcineurin/NFAT signalling and impaired autophagic response upon starvation. Pharmacologic miRNA inhibition by antagomir injection rescues cardiac hypertrophy and heart failure in mice, offering a possible therapeutic approach for cardiac failure.
Abstract 208: Farnesylation-Dependent Fibrosis in the Aged Murine Heart
Authors: Cieslik Katarzyna A , Entman Mark L , Trial JoAnn , Taffet George E
Published: July 2021

Mesenchymal stem cells (MSC) derived from the aged murine heart express reduced levels of the multipotency marker Nanog. These CD44
+
CD45
-
MSC inappropriately differentiate into fibroblasts characterized by markedly enhanced basal collagen expression. While collagen synthesis in cardiac fibroblasts derived from young mice is driven by TGF-β1, paradoxically, fibroblasts derived from the aged heart displayed reduced TGF-β1 responsiveness and decreased TGF-β receptor expression (ALK5). To resolve this apparent contradiction we looked for other pathways leading to collagen expression. We found that circulating insulin levels in aged mice were elevated ∼2.5 fold when compared to young animals. We hypothesized that increased circulating insulin levels drive MSC differentiation and caused upregulation of procollagen expression in cardiac fibroblasts. Our
in vitro
experiment confirmed that insulin at a pathophysiological concentration (1 nM) increased procollagen type I expression only in fibroblasts isolated from old hearts. As insulin can signal through substrate prenylation, cardiac fibroblasts isolated from aged mice had ∼2 fold greater basal activity of farnesyltransferase (FTase, enzyme that transfers the prenyl chain) than fibroblasts isolated from young mice. FTase activity was further increased when fibroblasts isolated from aged hearts were stimulated with insulin. Cells derived from 3 month old mice did not upregulate FTase activity in response to insulin. Basal expression of FTase regulatory subunit (FNTA) was also upregulated by 3 fold in aged fibroblasts and expression of catalytic subunit (FTNB) was increased with insulin stimulation. FTase inhibitor reduced procollagen synthesis in aged fibroblasts.

Here we present new data linking insulin-dependent upregulation of collagen type I synthesis via increased farnesylation in the aging heart.
Abstract 21: Stiffness-Dependent Notch1 Activation Regulates Cardiogenic Differentiation of Cardiac Progenitor Cells
Authors: Boopathy Archana V , Salaita Khalid , Davis Michael E
Published: July 2021

Cardiac progenitor cells (CPCs) are multipotent, self-renewing cells that can regenerate the myocardium and improve cardiac function in animal models of MI by cardiogenic differentiation. However, limited survival of stem/progenitor cells, myocardial scarring and fibrosis inhibit cardiac regeneration. Notch signaling promotes early cardiac development, cardiomyocyte survival and cardiac gene expression in circulating endothelial progenitor cells, mesenchymal stem cells and CPCs. As misregulation of Notch signaling during development is lethal due to cardiovascular defects, activating this critical pathway during cell transplantation could improve the efficacy of stem cell therapy. We investigated whether self-assembling peptide nanofiber hydrogels can be used to activate Notch1 signaling.

The 16 amino acid self-assembling scaffold (RAD) was modified with a 20 amino acid peptide mimicking the active site of Notch1 ligand, Jagged1 (RJAG) or with the corresponding scrambled peptide (RSCR). To determine whether scaffold stiffness regulates Notch1 activation, CHO cells with Notch1 responsive YFP expression were cultured in scaffolds of 1-3% w/v in presence of RSCR or RJAG at a 1:10 ligand: scaffold ratio in 3D. Presence of the RJAG peptide (p<0.01) and % concentration of the scaffold (p<0.01) increased Notch1 activation significantly (n=5) indicating that RJAG mediated Notch1 activation in 3D is scaffold stiffness-dependent. Therefore, CPCs were cultured within 3D scaffolds (1-3% w/v; empty, scaffold +RJAG or RSCR) and cardiogenic gene expression was determined by qPCR. An increase in expression of early endothelial (Flk1, Flt1, vWF) and smooth muscle (sm22α, sm αactin) genes was observed in CPCs cultured in 3D scaffolds containing RJAG but not when cultured in 2D. These data show that Notch1 activation is dependent on ligand density and scaffold stiffness. Delivery of CPCs in JAG1 containing self-assembling scaffolds could be used to enhance therapeutic angiogenesis and improve cardiac function following myocardial infarction.
Abstract 213: Rapid Activation of G-Protein Coupled Estrogen Receptor 1 Protects the Heart Against Ischemia-Reperfusion Injury by Inhibiting the mPTP Opening via PKC/AKT/ERK/GSK-3b Pathways
Authors: Bopassa Jean Chrisostome , Lu Rong , Singh Harpreet , Zilberstein Netanel F , Olde Bjorn , Krust Andree , Leeb-Lundberg L M Fredrik , Toro Ligia , Stefani Enrico
Published: July 2021

Introduction: Estrogen effect can be mediated by three receptors: Classical estrogen receptors: alpha (ERa) and beta (ERb), and recently identified G protein-coupled estrogen receptor1 (GPER1).

Hypothesis: We investigated the role of ERa, ERb and GPER1 in mediating rapid estrogen-induced cardioprotection in male mice hearts subjected to ischemia/reperfusion using wild type (WT) and gene specific knockout animals.

Methods: Isolated hearts from wild type (WT: C57BL/6NCrL), ERa-/-, ERb-/- and GPER1-/- were perfused using Langendorff apparatus with Krebs Henseleit buffer (control) or with the addition of estrogen (40 nM). Hearts were subjected to 18 min global ischemia followed by 60 min reperfusion. Cardiac function was recorded during the entire experiment and myocardial infarct size measured by TTC staining at the end of the reperfusion. Mitochondria calcium retention capacity (CRC) required to induce the mitochondrial permeability transition pore (mPTP) opening was assessed after 10 min reperfusion. Protein levels were measured by Western Blot in whole heart lysates after 5 min treatment just before ischemia, and after 10 min reperfusion. LY294002, U0126 and Chelerythrin-Cl were used as inhibitor of PI-3K/Akt, MAPK/ERK and PKC translocation, respectively.

Results: In WT, ERa-/- and ERb-/-, estrogen treatment significantly improved cardiac functional recovery, reduced infarct size and improved mitochondrial CRC. However, estrogen effects were completely absent in GPER1-/-. Estrogen treatment during 5 min before ischemia induced up-regulation of Akt, GSK-3b, and ERK1/2 phosphorylation in WT mouse as compared with control but not in GPER1-/-. However, after 10 min reperfusion estrogen effect was still oserved on GSK-3b, but not on Akt and ERK1/2. Chelerythrin-Cl prevented estrogen-induced cardioprotection effect and U126 abolished estrogen effect on mitochondrial CRC while
LY294002
could not prevent estrogen effect on GSK-3b observed in WT. P<0.05 and n=3-6.

Conclusion: Rapid activation of GPER1 induces cardioprotection effect against ischemia/reperfusion injury. Estrogen effects through GPER1 are associated with phosphorylation of Akt, GSK-3b and ERK1/2, translocation of PKC, and inhibition of the mPTP opening.
Abstract 214: Cbl Inhibition Improves Cardiac Function and Survival in Response to Myocardial Ischemia
Authors: Rafiq Khadija , Kolpakov Mikhail , Seqqat Rachid , Guo Jianfen , Guo Xinji , Sanjay Archana , Houser Steven R , Sabri Abdelkarim
Published: July 2021

Introduction and Hypothesis:
Proteasome degradation of ubiquitin-targeted proteins plays an important role in the development of cardiomyopathy and heart failure. The mechanisms that control proteasome function and protein ubiquitination and turnover remain poorly defined. The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets tyrosine kinases, resulting in their ubiquitination and down-regulation. Although Cbl is a crucial signaling molecule in many cells, the role of Cbl in the heart is unknown.

Methods and Results:
We show increased Cbl expression in human failing hearts and in response to pathological stress stimuli in mice. Cbl deficient mice demonstrated a more robust functional recovery after ischemia reperfusion injury, as well as significantly reduced myocyte apoptosis and epidermal growth factor receptor and focal adhesion (FA) protein ubiquitination and downregulation. Cbl Knockdown in-vitro protected cardiomyocytes against H2O2-induced apoptosis. Cbl deficient mice also show greater functional recovery after myocardial ischemia. This cardioprotective effect of Cbl deletion can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of vascular endothelial growth factor-a.

Conclusions:

Cbl activation regulates myocyte apoptosis and angiogenesis and contributes to adverse cardiac remodeling post-MI. These findings point to Cbl as a potential therapeutic target for the maintenance of cardiac function and remodeling post-MI.
Abstract 218: PI3Kγ Regulates Age-Dependent Cardiac Hypertrophy Through Kinase-Independent GSK-3-PP2A Axis
Authors: Naga Prasad Sathyamangla V , Mohan Maradumane L , Martelli Elizabeth E , Gupta Manveen K , Vasudevan Neelakantan T
Published: July 2021

Activation of phosphoinositide 3-kinase α (PI3Kα) by Receptor Tyrosine Kinase (RTK) or PI3Kγ by G-protein coupled receptor (GPCR) inhibits glycogen synthase kinase-3 (GSK-3) via protein kinase B (Akt). We show that in addition to promoting GSK-3 phosphorylation through Akt, PI3Kγ in parallel suppresses PP2A dependent GSK-3 dephosphorylation. This is evidenced by accelerated GSK-3 dephosphorylation in PI3Kγ knock out (PI3Kγ-KO) mice downstream of RTK-PI3Kα-Akt axis despite robust Akt activation by insulin. Confocal microscopy and immunoblotting show marked reduction of steady state GSK-3 phosphorylation in PI3Kγ-KO compared to littermate controls. Assessment of GSK-3 dephosphorylating enzyme protein phosphatase 2A (PP2A) showed significant elevation in PP2A and GSK-associated phosphatase activity in PI3Kγ-KO mice compared to controls. Mechanistically, we found that elevated PP2A activity in PI3Kγ-KO was due to PP2A methylation mediated by elevated PP2A methyl transferase (PPMT-1) activity. Consistent with the elevated anti-hypertrophic GSK-3 activity, we observed reduced heart size in PI3Kγ-KO mice at 6, 12, and 18 months compared to age matched littermate controls. To test in vivo whether PI3Kγ activity regulates cardiac GSK-3 function through PP2A, we bred transgenic mice with cardiac overexpression of inactive PI3Kγ (PI3Kγ
inact
) with PI3Kγ-KO mice. Surprisingly, cardiac overexpression of PI3Kγ
inact
transgene in PI3Kγ-KO background completely normalized cardiac PPMT-1 activity resulting in reduced PP2A activity and increased GSK-3 phosphorylation. Expression of PI3Kγ
inact
transgene in PI3Kγ-KO resulted in normalization of heart size compared to PI3Kγ-KO littermates consistent with the increased GSK-3 phosphorylation and consequent inhibition of GSK-3 activity suggesting a novel kinase independent role of PI3Kγ downstream of growth factor receptor in regulating cardiac growth with age.
Abstract 222: Combination of Blood Cardioplegic and Strategic Reperfusion Period Leucodepletion During Cardiopulmonary Bypass Preserves the Cardiac and Pulmonary Function: A Prospective Randomized Controlled Study
Authors: Chen Qiang , Sheikh Amir , Sheppard Stuart , Gibbs Roslyn , Smith David , Haw Marcus
Published: July 2021

Objectives:
Leucocytes activation during cardiopulmonary bypass (CPB) contributes to postoperative organ dysfunction. We compared the outcomes of various leucodepletion strategies in patients undergoing coronary artery bypass grafting (CABG).

Methods:
One hundred-twenty low-risk patients undergoing first time CABG were prospectively randomized to six groups: 1 non-leucodepletion arterial filtration; 2 continuous arterial leucodepletion; 3 reperfusion period leucodepletion; 4 leucodepletion of blood cardioplegia; 5 combination of continuous arterial and blood cardioplegic leucodepletion; 6 combination of blood cardioplegic and reperfusion period leucodepletion. Blood samples were taken 5 min before CPB, 5 and 30 min on CPB, 5, 60 min after aortic X-clamp removal and 6 h post-CPB. Activated leucocytes were identified with Nitroblue Tetrazolium staining. Exhaled nitro oxide (NO) was measured pre- and post-CPB using real-time chemiluminescense analyzer. Respiratory index (alveolar-arterial oxygenation index, AaOI) was calculated 5 min before CPB and 5 min on CPB, 1, 2, 4, 8 and 18 h post-CPB.

Results:
Activated white cell counts were similar before CPB in all groups, but reduced significantly 5, 60 min following aortic X-clamp removal, and 6 h post-CPB in groups 2, 3, 5 and 6, compared with group 1 (ANOVA p=0.02). Post-CPB, NO increased in all groups (p<0.05) except in Group 6 (3.17±0.64 vs. 3.63±0.79 ppb/s, p=0.22). Group 6 exhibited a lower AaOI than group 1 over all time points post-CPB (ANOVA, p=0.02). Postoperatively, there was a significant reduction in inotropic requirement, ventilation time and atrial fibrillation in group 6 (p=0.045, 0.04 and 0.043, respectively). There was no difference in chest drain output, length of ICU and hospital stay among all groups.

Conclusions:
Combination of blood cardioplegic and reperfusion period leucodepletion appeared to be most effective in attenuating activated leucocytes during CPB, which was associated with better preservation of cardiac and pulmonary function compared with other leucodepletion strategies.
Abstract 226: Mutual Interactive Factors β-Catenin and Oct4 Regulate Mesp1 Transcription
Authors: Li Yongqing , Schwartz Robert , Liu Yu
Published: July 2021

The b-HLH family protein Mesp1 sits on the top of the regulatory hierarchy of factors that govern cardiac specification. Ectopic expression of Mesp1 drives precocious appearance of cardiac progenitors, while ablation of Mesp1 and its close genetic relative Mesp2 completely blocked the emergence of heart in mouse embryos. Despite its central importance in cardiac specification, the regulation of Mesp1 is largely unknown. In this study, we show several lines of evidences supporting that beta-catenin and Oct4 cooperate in regulating Mesp1 induction. (1) A conserved TCF/Lef-Oct4 composite binding site is identified near the transcription start site of Mesp1. (2) beta-catenin and Oct4 each activates a Mesp1-Luciferase reporter, with beta-catenin being more potent; while together, beta-catenin and Oct4 synergize in activating the Mesp1-Luciferase reporter. Point mutations within the TCF/Lef-Oct4 binding site impair reporter activation by beta-catenin. (3) beta-catenin and Oct4 individually binds to an oligo covering the putative TCF/Lef-Oct4 binging site on Mesp1 promoter, and together they form a ternary complex, as detected by electrophoresis mobility shift assays. (4) Mesp1 induction is absent in cells with null alleles of beta-catenin, or conditional null alleles of Oct4. (5) While a 6K promoter of Mesp1 effectively drives expression of a lacZ reporter at E7.5, mutants affecting the TCF/Lef-Oct4 binding site fail to do so, in transient transgenic assays. Together, our results show that the cooperation between beta-catenin and Oct4, for Mesp1 transcription and cardiac specification substantiate a novel regulatory mechanism governing cell fate determination and cardiac development, which also explain the mechanism of well-established roles of each factor in mesoderm formation and heart development. Such knowledge may be used to establish new technology to generate cardiac cells for therapeutic purposes.
Abstract 248: Baseline Cognitive Impairment a Predictor of Mortality in Cardiovascular Disease
Authors: Lassaletta Antonio D , Sellke Frank W , Cohen Ronald A
Published: July 2021

Objective:
Few longitudinal studies have examined outcomes of patients with cardiovascular disease (CVD) in relation to earlier, baseline cognitive function, or examined the relationship between specific cognitive domains and mortality. This study attempted to clarify the relationship between cognitive function and long-term outcomes, particularly mortality, in patients with cardiovascular disease.

Methods:
One hundred seventy seven participants between 55 and 85 years of age with an established history CVD were recruited from healthcare institutions. A comprehensive neurophysiological test battery was administered to all patients at baseline assessing 4 cognitive domains: language, visual-spatial, memory, and attention-executive function. Composite scores were calculated by averaging z-scores of all component measures. At 6-year the follow-up, of the 136 patients who were still enrolled, the 116 who were still alive were contacted by phone for assessment of level of independent living, and subjective assessment of cognitive function, and quality of life.Results were corrected for age.

Results:
At follow-up, participants who were deceased had significantly lower baseline attention-executive function (M=-0.51, SD=0.78 vs. M=0.06, SD=0.54 in survivors, p < 0.01). Poorer memory performance at baseline was associated with a greater degree of cognitive dysfunction at follow-up (p = 0.085). No significant relationships were found between baseline cognitive performance and independent living status or quality of life.

Conclusion:
Impaired executive function was predictive of poorer outcomes, specifically, 6-year mortality. This finding reinforces the frontal nature of vascular cognitive impairments, especially in regard to the long term sequelae of cognitive dysfunction in patients with CVD. The higher mortality observed in patients with baseline executive domain impairment may be due to behaviors such as impaired self-care, treatment adherence and rehab participation or functional impairments that exacerbate health problems, resulting in death. Patients with cardiovascular disease and executive function impairment should be identified and measures taken to address these potential problems.
Abstract 249: Sigma-1 Receptor Agonist Ameliorates Mitochondrial ATP Production and Apoptosis in Cardiac Myocytes
Authors: Tagashira Hideaki , Shioda Norifumi , Bhuiyan Md. Shenuarin , Fukunaga Kohji
Published: July 2021

Objective:
Selective serotonin reuptake inhibitors (SSRIs) are known to reduce post-myocardial infarction (MI)-induced morbidity and mortality. However, the molecular mechanism underlying SSRI-induced cardioprotection remains unclear. We previous reported that fluvoxamine with high affinity for sigma-1 receptor ameliorates cardiac hypertrophy and dysfunction via sigma-1 receptor stimulation. In non-cardiomyocytes, sigma-1 receptor interacts with IP
3
receptor (IP
3
R), which may promote Ca
2+
transport to mitochondria. We here investigated the role of sigma-1 receptor for sarcoplasmic reticulum (SR)-mitochondrial Ca
2+
signaling in neonatal rat ventricular cardiomyocytes.

Methods:
Cultured cardiomyocytes were treated with angiotensin II (Ang II) during 72 hr followed by fluvoxamine and/or NE-100 treatment during the last 24 hr. Then, we investigated intracellular localization of sigma-1 receptor and IP
3
R. We also measured phenylephrine (PE)-induced mitochondrial Ca
2+
and cytosolic Ca
2+
mobilization and ATP content in Ang II-treated cardiomyocytes with or without fluvoxamine treatments.

Results:
Ang II stimulation for 72 hr elicited cardiomyocyte hypertrophy, downregulation of sigma-1 receptor expression and declined PE-induced Ca
2+
mobilization into cytosol and mitochondria. Fluvoxamine treatments restored sigma-1 receptor expression and PE-induced Ca
2+
mobilization into mitochondria. Moreover, fluvoxamine treatment completely restored Ang II-induced apoptosis. We also confirmed in vivo that fluvoxamine treatment rescue transverse aortic constriction-induced cardiac dysfunction and the reduced ATP concentration.

Conclusions:
These results suggested that fluvoxamine rescue cardiomyocytes from AngII-induced cardiac myocyte apoptosis through enhancement of SR-mitochondria Ca
2+
transport and mitochondrial ATP production via sigma-1 receptor stimulation.
Abstract 255: A Normokalemic Nondepolarizing Cardioplegia Minimizes Myocardial Injury and Improves Cardiac Function in a Large Animal Model of Donation After Cardiocirculatory Death
Authors: White Christopher W , Ali Ayyaz , Xiang Bo , Colah Simon , Mundt Paul , Arora Rakesh , Lee Trevor , Large Steven , Tian Ganghong , Freed Darren
Published: July 2021

Background:
Procurement of donor hearts is typically preceded by delivery of a cold hyperkalemic cardioplegia (CP) that achieves arrest by depolarization of the membrane potential. This method has no intrinsic cardioprotective properties and may exacerbate ischemia-reperfusion (IR) injury. A tepid adenosine-lidocaine (AL) non-depolarizing CP has been shown to limit IR injury and improve myocardial functional recovery in a small animal model of orthotopic heart transplantation. We sought to determine the impact of AL CP on myocardial injury and function, in a porcine model of donation after cardio-circulatory death (DCD).

Methods:
Female Yorkshire pigs (60 kg) were subjected to a hypoxic cardiac arrest. Following a 15-minute standoff period donor hearts were procured, resuscitated ex-vivo, and transplanted into a recipient animal. Hearts in group 1 (N=7) were protected with a cold blood-Plegisol CP, perfused with a blood-crystalloid solution during ex-vivo assessment, and arrested with a cold blood-Plegisol CP prior to transplantation. Hearts in group 2 (N=5) were protected with a tepid AL-STEEN CP and perfused with a blood-STEEN solution in a continuous fashion during ex-vivo assessment and transplantation.

Results:
Animals in group 2 had a significantly longer ex-vivo perfusion time compared to those in group 1 (174±29 vs. 135±45 min, p<0.05), yet they demonstrated significantly less weight gain (14±4 vs. 25±8 grams/hour, p<0.05). Further, animals in group 2 exhibited less troponin I release during ex-vivo perfusion and had significantly better diastolic function following wean from cardiopulmonary bypass (left ventricular end-diastolic pressure volume relationship 0.115±0.052 vs. 0.508±0.223, p<0.05). However, there was no significant difference in level of calpain activity between groups (171.3±68.6 vs. 140.12±50.6, p=0.36).

Conclusion:
A tepid non-depolarizing AL CP results in less myocardial edema during ex-vivo perfusion, minimizes myocardial injury, and improves diastolic function following transplantation of DCD hearts, but fails to eliminate the activation of calcium-dependent proteases. Further studies should investigate if a calpain inhibitor delivered at reperfusion could further minimize IR injury in DCD hearts.
Abstract 256: Dysfunctional Cardiac Stem Cell Niches Condition the Aging of the Heart
Authors: Sanada Fumihiro , Zheng Hanqiao , Cappetta Donato , Mangano Emily , Kim Junghyun , Arranto Christian , Ide-Iwata Noriko , Pesapane Ada , Ricciardi Mario , Gurusamy Narasimman , Goichberg Polina , Rota Marcello , Hosoda Toru , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

The objective of this study was to determine whether aging of the heart is characterized by alterations in the cellular composition of cardiac niches and defective cardiomyogenesis. By employing a morphometric approach in which the three-dimensional structure of each niche was evaluated, we examined 40 and 20 cardiac niches in young and old Fischer 344 rats, respectively. Although a niche can include a single stem cell, clusters composed of three or more cardiac stem cells (CSCs) and early lineage committed cells (LCCs) were included in the analysis. LCCs expressed the stem cell antigen c-kit in combination with the marker of cardiac commitment GATA4. The average volume of the niches did not vary with age. Surprisingly, the number of lineage-negative CSCs increased from 4 to 28 months. Young niches contained 6 CSCs while old niches showed 10 CSCs. However, the fraction of CSCs positive for the senescence-associated marker p16
INK4a
increased 14-fold with age, from 6% to 85%. Corresponding values for LCCs were 8% and 82%. Apoptosis of CSCs-LCCs increased with age and was restricted to p16
INK4a
-positive-cells. However, the fraction of senescent CSCs-LCCs undergoing apoptosis decreased 3.5-fold with age: apoptosis was detected in 7% and 2% of p16
INK4a
positive CSCs-LCCs of young and old animals, respectively resulting in the accumulation of senescent cells.Freshly isolated CSCs proliferated in response to growth stimuli but this effect was attenuated in old cells. This finding was consistent with the decreased expression of the insulin-like growth factor receptor 1 (IGF-1R) in senescent CSCs. Collectively, the accumulation of senescent CSCs and the poor response of p16
INK4a-
negative CSCs to growth stimuli determined the formation of dysfunctional niches. Abnormal niche turnover led to the generation of rapidly aging cardiomyocytes. In conclusion, the cellular composition of the niches varies with age. The decrease in the number of functionally-competent CSCs-LCCs together with the accumulation of senescent CSCs-LCCs limits the ability of CSC niches to repopulate the senescent ventricular myocardium.
Abstract 258: Human 450delACryAB Overexpression Associated with Protein Aggregation Uncovers a Novel Mechanism for Inheritable Myofibrillar-Sparing Cardiomyopathy in Mice
Authors: Zhang Huali , Liu Qiang , Limphong Pattraranee , Wende Adam R , Wang Xiaohui , Zhang Xiuquan , Pratt Gregory W , Christians Elisabeth , Benjamin Ivor J
Published: July 2021

Desmin-related cardiomyopathy (DRC) belongs to the family of myofibrillar diseases characterized by the presence of toxic desmin-positive aggregates and degeneration at Z-disk structures. DRC has been linked to the mutations in the desmin, αBcrystallin (CryAB), or myotilin genes. Several human CryAB mutations cause multisystem disorders and experimental models of R120GCryAB in transgenic mice strikingly recapitulate similar phenotype observed in patients. In this context, we have generated the 450delACryAB linked to inheritable cataracts in neonatal rat cardiomyocytes and mice hearts. 450delACryAB formed aggregates or aggresomes, and recruited HSP25 into the insoluble fraction in the cardiomyocytes. However, heart-specific 450delA CryAB overexpression neither causes desmin-related cardiomyopathy nor exacerbates pressure overload-induced hypertrophy. To our knowledge, this is the first report of compensation to protein aggregates formation abrogates the development of cardiomyopathy and suggests possible insights into disease-specific susceptibility and resistance. We found that there are several different characteristics between 450delACryAB and R120GCryAB. First, 450delACryAB formed much less soluble oligomers than R120GCryAB in cardiomyocytes, and translocated almost into the insoluble fraction when overexpressing in the hearts. Secondly, 450delACryAB aggregates displayed small pattern without either disturbance of desmin striated network or sequestration of desmin into the insoluble fraction, although 450delACryAB interacts with desmin in the heart. Thirdly, doxycycline treatment did not suppress 450delACryAB aggregates. Nonetheless, 450delACryAB aggregates caused the accumulation of ubiquitinated proteins and subsequently upregulated autophagy activity in mice heart, which is similar to R120GCryAB aggregates. The interaction of 450delACryAB with AKT, p62 and Beclin 1 might contribute to impaired UPS dysfunction and activation of autophagy. The findings reported here suggest that such differences contribute to the variable penetrance and pathogenesis in humans.
Abstract 26: Myocardial Phosphodiesterase-2A Is Upregulated in Human and Experimental Heart Failure and Blunts Cardiac β-Adrenergic Inotropic Responsiveness
Authors: Vettel Christiane , Mehel Hind , Emons Julius , Wittkoepper Katrin , Seppelt Danilo , Lutz Susanne , Nikolaev Viacheslav O , Sosalla Samuel , Zimmermann Wolfram H , Vandecasteele Grégoire , Fischmeister Rodolphe , El-Armouche Ali
Published: July 2021

Augmented cGMP- and diminished cAMP-signaling within cardiomyocytes is characteristic for failing hearts. Cyclic nucleotide phosphodiesterases (PDEs) comprise a family of cyclic-nucleotide hydrolyzing enzymes, controlling cAMP and cGMP levels. Among them the PDE-2A isoform has the unique property to be stimulated by cGMP but primarily hydrolyzing cAMP. This appears to mediate a negative cross-talk between both signaling pathways. However, a potential role for PDE-2A in the failing heart has not been addressed yet. Here we show that PDE-2A protein levels were ∼2-fold higher in failing human hearts as well as in a large animal heart failure model from dog hearts subjected to rapid-pacing (n≥6, p<0.05). Intriguingly, PDE-2A protein levels were normal in hypertrophied hearts from patients with preserved cardiac function who underwent aortic valve replacement. Chronic beta-adrenergic stimulation by catecholamine infusions enhanced cAMP hydrolyzing activity of PDE-2A by four-fold (n≥6, p<0.05) in rat hearts in vivo and in isolated cardiomyocytes (measured by radioimmunoassay and FRET-based sensors, respectively) and correlated with blunted beta-adrenergic responsiveness. Consistent with this observation, overexpressed PDE-2A, which localized to the sarcomeric Z-line, blunted the rise in cAMP by 70% (n≥6, p<0.05) and abolished the positive inotropic effect after acute beta-adrenergic stimulation by 70% (n≥6, p<0.05) in isolated cardiomyocytes. Notably, those cardiomyocytes also showed marked protection from norepinephrine-induced hypertrophic responses, e. g. 40% less increase in cell surface area (n≥10, p<0.05). In summary, PDE-2A is markedly upregulated in human and experimental failing hearts. This may constitute an important defense mechanism during cardiac stress, by antagonizing the cAMP-mediated toxic effects. Thus, activating myocardial PDE-2A may represent a new intracellular anti-adrenergic therapeutic strategy in heart failure.
Abstract 260: Time Course Observation of Left Ventricular Hypertrophy and Regression Induced by an Improved Technique
Authors: Zhang Xiu , Li Ling , Szucsik Amanda , Javan Hadi , Deng Yubin , Zhang Ren , Selzman Craig
Published: July 2021

In order to study the left ventricular hypertrophy (LVH) and its regression, we improved the surgical technique for producing LV pressure overload and its reversing mouse model using the titanium clip placement for transverse aortic banding (AB) and removal for debanding (DB). A time course study on heart function and histology proved the importance of the regression time.

Materials and methods:
The technique was applying titanium clip for AB and removing for DB with minimal invasion. A total of 95 male C57BL6 mice weighing 20-30 g were used in this study. Experimental groups included: control (n=11), AB 3 (11), 4 (12), and 6 (12) wks, DB at 3 (12), 4 (11), and 6 (12) wks of AB for 1 wk. Carotid arteries velocity were measured to evaluate the constrictive level. Heart/body weight ratio, cardiac wall thickness from echocardiography, LV pressure from catheterization and heart histology studies were used to evaluate the pressure overload and hypertrophy.

Results:
The echocardiographic and histology results indicated that AB induce significent LVH which was a concentric hypertrophy at AB 3 wks and then progressively become a dilated hypertrophy at AB 6 wks. The cardiac function also indicated a stable ejection fraction before 3 wks of AB and then gradually declined at AB 6 wks. After 6 wks AB, the enlargement in LV dimensions becomes significant. When deband at 3 and 4 wks for one week, there were significant regression of hypertrophy but the regression is limited after AB 6 wks and the evidences of lung congestion were apparent. In catheterizaiton, the LV systolic pressure were markedly elevated in AB mice but these pressure elevations became less at AB 6 wks and the LV end-diastolic pressure (LVEDP) at AB 6 wks was elevated significantly. After DB at AB 3 and 4 wks, the pressure parameters retriced close to normal but LVEPD elevation retreat is limited in the DB 6 wks group.

Conclusion:
With applying and removing titanium clip to induce pressure overload and reversing can provide a relatively simple, effecive, consistent and minimal invasive methods to establish the mouse model for studying LVH and its regression. Our data indicate that AB induced mouse LVH could be retriced close to normal when remove the constriction at the concentric LVH phase but the regression is limited in dilated LVH.
Abstract 266: Autophagy Reverses Intramuscular Lipid Accumulation in Myocytes
Authors: Lam Truong N , Harmancey Romain , Taegtmeyer Heinrich
Published: July 2021

Autophagy, or “self-eating” of the cell, is a tightly regulated “housekeeping” process involved in the degradation and recycling of protein aggregates and damaged organelles. In the failing heart, autophagy has been shown to be an adaptive response. We have previously demonstrated that a common feature of non-ischemic heart failure is lipid accumulation, which in excess can mediate cardiac dysfunction via lipotoxicity. Since autophagy mediates the clearance of lipids through a specific process, known as lipophagy, in hepatocytes, we wondered whether the activation of autophagy in the failing heart protects myocyte from lipotoxicity. To test this hypothesis, L6 myocytes were incubated over a period of 6 days with long chain fatty acids, either 0.5mM or 1.0mM fatty acids (equimolar mixture of oleate and palmitate). On the sixth day of treatment, an autophagic inhibitor (bafilomycin A1, 200nM) or autophagic activator (rapamycin, 1uM) was added to the cell culture medium for 24 hours. Intracellular triglyceride (TG) accumulation was measured in the cells using enzyme quantification assay as well as Oil Red O stain. Immunoblotting was also performed on protein markers to confirm autophagic flux (LC3 and P62) and cell death (Caspase3) in the myocytes. The results indicated that increasing concentrations of fatty acids gradually increased intracellular TG accumulation. Inhibition of autophagy using bafilomycin increased this effect whereas activation of autophagy using rapamycin reduced lipid accumulation in the L6 myocytes. Moreover, fatty acid mediated cell death was increased when autophagy is inhibited. We conclude that autophagy promotes the clearance of lipids from cultured myocytes. These findings suggest that lipophagy may be a protective mechanism in the myocyte through the decrease and reversal of intracellular lipid accumulation.
Abstract 27: The Transcription Cofactor Eya4 Mediates the Development of Acquired Cardiac Hypertrophy
Authors: Williams Tatjana , Schoenberger Jost , Hundertmark Moritz , Nordbeck Peter , Voll Sabine , Czolbe Martin , Pekarek Vera , Ritter Oliver
Published: July 2021

Introduction:
Eyes absent 4 (Eya4) is a transcription cofactor involved in a number of cellular and developmental processes. We have previously shown that a mutation in Eya4 (E193) leads to late-onset familial dilated cardiomyopathy and heart failure. A precise role for Eya4 in the myocardium has not yet been identified. It appears to regulate the cyclin dependent kinase inhibitor p27
kip1
(p27), a protein shown to regulate hypertrophic responses in the adult cardiomyocyte. This study was aimed to explore the role of Eya4 in induced cardiac hypertrophy.

Methods and results:
We generated transgenic mice with cardiac-specific overexpression of HA-tagged Eya4 or E193 to elucidate the function of these proteins in the development of heart failure
in vivo
. These and wildtype littermates were challenged with angiotensin II (ATII) or subjected to transaortic constriction (TAC). Magnetic resonance imaging to visualize cardiac structures in detail showed that in response to sustained ATII stimulation and TAC, Eya4 mice exhibited a phenotype with significantly increased parameters of hypertrophy compared to WT and E193 overexpressing animals as judged by increases in heart weight and LV free wall diameter, cross-sectional cell surface areas and fibrosis. MRI also showed mild cardiac hypertrophy in Eya4 transgenic mice already under baseline conditions in an age dependent fashion. Moreover, Eya4 overexpression induced a significant suppression of p27 protein expression and resulted in increased levels of phosphorylated histone deacetylase 2 (HDAC2). E193 overexpression induced age dependent wall thinning and ventricular dilation under baseline conditions with no obvious structural or functional defects. ATII or TAC induced significant changes in HW/BW ratio, IVS, fibrosis, hemodynamic and cell size measurements, albeit to a lesser extent than seen with Eya4 mice. p27 expression and pHDAC2 levels were only slightly altered.

Conclusion:
In summary, we have demonstrated a mutation in Eya4 to disturb cardiac physiology. We now provide evidence that Eya4 is also involved in forms of acquired heart disease. It seems to suppress p27, which leads to phosphorylation and activation of HDAC2 and results in the development of cardiac hypertrophy.
Abstract 280: Protection of Oral Hydrogen Water as an Antioxidant on Pulmonary Hypertension
Authors: He Bin , Zhang Yufeng , Kang Bo , Xiao Jian , Xie Bing , Wang Zhinong
Published: July 2021

Rationale:

This study was to explore protection of hydrogen as an antioxidant on monocrotaline (MCT)-induced pulmonary hypertension (PH). Instead of hydrogen-rich saline, we made new modification by using oral hydrogen water (HW), which is more convenient, less costly and longer time to be released.

Methods:

Thirty-six SD rats were equally randomized to three groups: SHAM group, MCT group and MCT+HW group. The PH model was established by intraperitoneal injection of MCT (80 mg/kg body weight). The mean pulmonary arterial pressure (mPAP) was measured two weeks after establishment of the model, ensuring that mPAP in the modeled rats was higher than that in SHAM group. Animals in MCT group were administered with ordinary drinking water, and those in MCT+HW group were administered with HW. Two weeks later, the animals were sacrificed, and the heart and lung tissue were removed for measurement of the ventricular weight, left ventricular+septum weight, and right ventricle weight (RV). The right ventricular hypertrophy index (RVHI) was calculated. Changes in the degree of damage to the lung tissue was observed and scored by HE staining. Changes of 3-nitrityrosine (3-nt) and Intercellular Adhesion Molecule-1 (ICAM-1) in the lung tissue were observed by immunohistochemistry.

Results:

By the end of the experiment at day 28, mPAP, RV and RVHI in MCT group were 44.6±7.2 mmHg, 0.438±0.051 g and 0.59±0.065 respectively, which were significant higher than those in SHAM group (
P
< 0.05). mPAP, RV and RVHI in MCT+HW group were 31.4±6.5 mmHg, 0.346±0.041 g and 0.45±0.048 respectively, which were significantly lower than those in MCT group (
P
< 0.05). In addition, pulmonary inflammatory response, 3-nt and ICAM-1 were increased significantly in MCT group and decreased significantly in MCT+HW group as compared with SHAM group (both
P
< 0.05).

Conclusions:

Oral hydrogen water has a significant protective effect on MCT-induced PH, which is more convenient to use than hydrogen-rich saline. And this effect is associated with the antioxidative ability of hydrogen and its action of reducing pulmonary inflammatory response.