bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P321: Oxidative Stress Induces DNA Double-Strand Breaks and Activates DNA Damage Responses in Vascular Smooth Muscle Cells: A Possible Mechanism for Atherosclerosis Progression
Authors: Ishida Takafumi , Ishida Mari , Tashiro Satoshi , Sakai Chiemi , Uchida Hitomi , Miyagawa Kiyoshi , Yoshizumi Masao , Kihara Yasuki
Published: July 2021

Backgrounds:
Oxidative stress is thought to be a pathogenic mediator of atherosclerosis. Oxidative stress induces DNA damage, and the unrepaired or improperly repaired DNA damage increases genomic instability, which cause cell death, senescence, or dysregulation of cellular functions. Pathogenesis of both Hutchinson-Gilford Progeria syndrome and Werner syndrome, which feature prominent atherosclerotic disease at young age, involves impaired DNA repair and the resultant genomic instability. The purpose of this study is to determine whether oxidative stress causes DNA damage in vascular smooth muscle cells (VSMC) and to elucidate the role of DNA damage responses in atherosclerosis and the fate of VSMC.

Methods and Results:
Immunoreactivity against gamma-H2AX, a sensitive marker for DNA double-strand breaks (DSBs), which is the most severe form of DNA damage, was increased in human atherosclerotic plaques, but not in the adjacent normal areas. gamma-H2AX staining was observed in almost same regions where 8-oxo-dG immunoreactivity, an oxidative modification of DNA, was observed. Apoptotic cells were abundant in atherosclerotic lesions, but not in normal areas. In cultured human aortic smooth muscle cells (HASM), 15 min incubation with H2O2 (100 microM) induced foci formation of gamma-H2AX in the nuclei. H2O2 activated various signaling molecules involved in DNA damage responses, including ATM, Chk2, DNA-PK and p53 in HASM. Some H2O2-induced DSBs persisted after 24 hours, at which point apoptosis was induced in 7.1 ± 1.3 % of HASM, as detected by TUNEL method. Knockdown experiments using siRNA revealed that ATM-, DNA-PK-, or Chk2-deficient VSMC were more resistant to H2O2-induced apoptosis.

Conclusions:
In summary, 1) DNA double-strand breaks were accumulated in human atherosclerotic plaques, 2) oxidative stress induced double-strand breaks and activation of DNA damage response in vascular smooth muscle cells, and 3) impairment of DNA damage responses modulated damage-induced cell fate such as apoptosis. Thus, DNA damage itself or alteration in DNA damage responses may be involved in the mechanisms for progression of atherosclerosis.
Abstract P324: Unhealthy Diet and Air Pollution Compromise Human Cardiovascular Cell Functions: A First Mechanistic Analysis
Authors: Ale-Agha Niloofar , Buechner Nicole , Sydlik Ulrich , Unfried Klaus , Altschmied Joachim , Haendeler Judith
Published: July 2021

Diet and pollution are environmental factors known to compromise ″healthy cardiovascular aging″. The molecular consequences of the permanent burden for aging of the cardiovascular system are unknown, since they have never been examined in primary, adult human cells. Therefore, this study investigates the impact of unhealthy diet on aging-related signaling pathways of human, primary cardiovascular cells and of airborne particles on human endothelial cells, as several studies demonstrated that ultrafine particles can enter the circulation and thus may interact with endothelial cells directly. Nutrition health reports have shown that the diet in industrialized countries contains more than 100 mg/dl low density lipoprotein (LDL) and a too high fraction of monosaccharides, especially fructose, which is metabolized insulin-independently. Both components have been shown to increase the risk for cardiovascular diseases. To simulate unhealthy diet we supplemented cell culture media of human, primary endothelial cells (EC), smooth muscle cells (SMC) and cardiomyocytes (CM) with 100 mg/dl LDL and replaced 1/3 of the glucose with fructose for one week. This treatment did not induce cell death in any of the cell types. However, we observed increased senescence, loss of endothelial nitric oxide synthase and increased nuclear localization of Foxo3 in EC, increased proliferation in SMC and hypertrophy in CM. With respect to pollution we have used ultrafine carbon black particles (ufCB), one of the major constituents of industrial and exhaust emissions, in concentrations our vessels are constantly exposed to. These concentrations of ufCB are non-toxic and non-inflammatory for EC. Despite these missing immediate effects, ufCB dramatically reduced the S-NO content, a marker for NO-bioavailability in EC and increased reactive oxgen species formation. As a consequence, ufCB dramatically increased senescence of EC after two weeks. Thus, unhealthy diet and a high burden of ultrafine carbon black nanoparticles, to which we are exposed every day, seem to induce a ″cardiovascular aging″ phenotype and can lead to severe cardiovascular diseases.
Abstract P328: β-Blockers Can Enhance Short-Term Receptor Sensitivity in Silico and in Vitro
Authors: Amanfu Robert , Saucerman Jeffrey
Published: July 2021

A key feature of heart failure is the chronic elevation of circulating catecholamines which desensitizes the β-adrenergic receptor signaling pathway, rendering patients incapable of increasing cardiac output in response to acute stress (eg. exercise). β-adrenergic receptor blockers (β-blockers) are commonly used to treat this condition, but precisely how they work is still controversial and poorly understood. Two opposing theories are that β-blockers work by 1) blocking the harmful consequences of chronic signaling or 2) paradoxically increasing receptor sensitivity. To investigate whether β-blockers function by increasing receptor sensitivity, we extended a published computational model of the β
1
-adrenergic receptor signaling pathway developed in our lab by using a ternary complex receptor module. This receptor model includes the spontaneous switching between the active and inactive conformations of the receptor that is crucial for accurate representation of β-blockers. Parameter values were determined from literature to model 11 agonists and 10 β-blockers. The new receptor model was validated against ligand binding studies and adenylyl cyclase assays from literature. Chronic and acute stresses were modeled as low and high (100X greater) concentrations of the agonist isoproterenol. Simulations indicate that when the β-blocker propranolol is present during chronic stress, cAMP levels are increased upon application of an acute stress, elevating intracellular calcium levels and contractility. This suggests that in addition to blocking the harmful consequences of chronic signaling, β-blockers may sensitize the response to acute stress. Preliminary data from calcium imaging experiments in isolated cardiac ventricular cells confirm this prediction. Simulations with a range of commonly used β-blockers indicate that individual drugs differ significantly in their ability to enhance receptor sensitivity. In addition, simulation of the Gly389Arg polymorphism suggests that the Arg389 variant has reduced sensitization to acute stress in the presence of propranolol. These simulations are a first step towards evaluating personalized β-blocker therapies with computational models.
Abstract P348: High Glucose Induces Smad Activation via the Transcriptional Coregulator P300 and Contributes to Cardiac Fibrosis and Hypertrophy
Authors: Connelly, Kim A , Ouzounian Maral , Advani Andrew , Advani Suzanne , Thai Kerri , Liu Peter P , Gilbert Richard E
Published: July 2021

Background:
Despite advances in the treatment of heart failure (HF), the mortality remains high, particularly in those individuals with diabetes mellitus. Activated transforming growth factor beta (TGF-ß) contributes to the pathogenesis of diabetic cardiomyopathy. We hypothesized that the transcriptional co-activator p300 regulates glucose induced activation of TGF-ß via acetylation of a specific Lysine residue (Lys19) in the Mad homology 1 domain of Smad 2, and that by inhibiting p300, TGF-ß activity will be reduced and heart failure ameliorated/prevented.

Methods:
p300 activity and Smad acetylation in normal glucose (5.6 mmol/L - NG) and high glucose (25 mmol/L - HG) media were assessed in H9c2 rat cardiomyoblasts. [H]3 proline incorporation was assessed in cardiac fibroblasts as a marker of collagen synthesis. The role of increased p300 activity was assessed in vitro by using a known p300 inhibitor, curcumin or siRNA directed at p300 and in vivo in a hemodynamically validated model of diabetic cardiomyopathy, the (mRen)2-27 transgenic rat.

Results:
H9c2 cells exposed to HG demonstrated increased p300 activity c/w NG media, that was reduced by p300 inhibition using curcumin or p300 siRNA (all p<0.01). Increased p300 activity in HG media increased [H]3 proline incorporation (p<0.05). This effect was attenuated by treatment with curcumin/p300 siRNA (p<0.01). Finally, H9c2 cells were stimulated, extracted protein was immunoprecipitated with Smad2, and lys19 acetylation assessed. Acetylation of the Lys19 was reduced in cells pre-incubated with the p300 inhibitor (p<0.05). To determine the functional significance of p300 inhibition, diabetic Ren-2 rats were randomised to receive either curcumin/vehicle for 6 weeks. Curcumin treated diabetic rats had reduced cardiac hypertrophy and improved chamber compliance when c/w untreated diabetic counterparts (all p<0.01).

Conclusions:
These findings demonstrate that high glucose increases activity of the transcriptional coregulator p300, acetylating Smad2 and promoting cardiac fibrosis and hypertrophy. Inhibition of p300 reduces cardiac hypertrophy and results in improved diastolic function. Modulation of the p300 may be a novel strategy to treat diabetes induced heart failure.
Abstract P351: Statins Prevent Expansive Remodeling in Arterialized Vein Grafts
Authors: Qiang Beiping , Toma Jonathan , Fujii Hiroko , Osherov Azriel , Nili Nafiseh , Fefer Paul , Samuel Michelle , Butany Jagdish , Leong-Poi Howard , Strauss Bradley H
Published: July 2021

Vein grafts (VG) account for more than 70% of conduits used in aortocoronary bypass surgery, but have high failure rates by 10 years. We have previously shown in an experimental model that VG undergo marked expansion by 4 weeks, which was followed by increased total LDL retention at 12 weeks. The objective of this study was to determine whether statin therapy would prevent these expansive remodelling changes.

Methods and Results
Reversed jugular vein-to-common carotid artery interposition graft were constructed in 24 cholesterol-fed (0.5%) rabbits. Rabbits were randomized to either control or atorvastatin (5 mg/kg/day) groups, starting two weeks prior to vein graft implantation and continuing until sacrifice at 1 or 12 weeks post-surgery. Ultrasound measurements of arterial luminal cross-sectional area (LCSA) were done at day 3 and at 4, 8 and 12 weeks post-surgery. Histomorphometric measurements were performed following sacrifice at 12 weeks. Atorvastatin treatment significantly decreased total plasma cholesterol levels at 12 weeks (6.70±4.19mmol/L vs placebo 38.65±10.55mmol/L, p<0.001). Atorvastatin significantly reduced expansive remodelling at all time points, including at 12 weeks (44.6±6.62mm
2
vs placebo 77.6±10.68mm
2
, p<0.001). Intimal CSA by histomorphometry was significantly reduced by atorvastatin at 12 weeks (5.59±2.19mm
2
vs placebo 9.57±2.43mm
2
, p<0.05). At 1 week, macrophage infiltration was significantly reduced (Ram11 positive intimal staining 0.018±0.019 vs 0.189±0.049, p<0.05). Atorvastatin treated samples at 1 week showed reduction in both MMP 2 activity (gelatin zymography) and metalloelastase activity (casein zymography).

Conclusion:
Atorvastatin prevents expansive remodelling in arterialized VG, likely through inhibition of macrophage infiltration and reduction of tissue proteolytic activity. This may be an important mechanism preventing VG atherosclerosis and late VG failure.
Abstract 106: A Novel Small Molecule Protects Against Myocardial Ischemia and Doxorubicin-Induced Cardiomyopathy Through Heat Shock Response and Proteostasis Regulation In Vivo
Authors: Sharma Sudhish , Deshmukh Savitha , Zampino Manuela , Walker Brandon , Kaushal Sunjay
Published: July 2021

Background:
A potentially new non-cell-based therapy is the systemic delivery of small molecules to revive or protect injured myocardium. By screening a drug library, we identified a novel small molecule, sk421, and tested its effectiveness in two myocardial injury models in vivo: a myocardial infarction model and doxorubicin (DOX)-induced cardiomyopathy model.

Results:
Sk421 significantly increased cellular viability of DOX-injured cardiomyocytes by preventing apoptosis in vitro, demonstrated by assays for MTT (p=0.042), annexin-V, and active caspase-3 (p=0.004). Sk421 increased the concentrations of the GATA4 and the heat shock proteins (HSPs) in cardiomyocytes (by 1.5-fold), and fibroblasts (by 1.2-fold), but was unable to rescue DOX-injured fibroblast cells depleted of HSPs. Sk421 maintained the total proteosome by a 3.5-fold inhibition of ubiquitinized GATA4 degradation, as shown by immunoprecipitation assays. Oral administration of sk421(4mg/kg) rescued DOX-injured mice by significantly preserving cardiac function, weight gain, cardiomyocyte lesion size and abundance, and by reducing overall mortality (20% in treated groups) compared to control DOX-injured mice (80% in untreated groups, p<0.03). The levels of GATA4 and HSPs were increased in the sk421-rescued mice. In the MI model, (n=5/group), sk421 reduced myocardial infarct size, and protected heart function evaluated by ejection fraction 45.8 ± 2.4 vs. 34.8 ± 3.3, p=0.005 (day7), and 44.3 ± 2.8 vs. 34.4 ± 4.1, p=0.011 (day28). Sk421 treatment of MI rats resulted in a higher fraction of viable tissue (within the LV (70.20 ± 0.5024, n=3) as compared to only MI group (56.38 ± 3.879%, n=3) and the infiltration of CD68+ cells was found to be more in the infarct region of LV of MI group rats as compared to MI+CEL. Quantitative analysis showed a significantly higher proportion of TUNEL positive cells and mRNA levels of IL-1β, IL-6, TNF-α and MMP2 in the myocardium of MI rats compared to MI+SK421 rats.

Conclusion:
This study demonstrates the broad therapeutic benefit of sk421 in animal model of myocardial injury by mechanism of inducing the heat shock response and preserving GATA levels. The ease of oral delivery makes sk421 a potentially clinically useful adjunct for myocardial preservation.
Abstract 110: Noncoding RNAs: A Possible "Linc" to Atrial Fibrillation
Authors: Gore Shamone R , Hsu Jeffery , Hanna Peter , Van Wagoner David R , Barnard John , Chung Mina K , Smith Jonathan D
Published: July 2021

Atrial fibrillation (AF), the most common cardiac arrhythmia, is associated with a 2-fold increased risk of mortality and a ~5- fold increased risk of stroke. Genome wide association studies have identified a locus on chromosome 4q25 that has the strongest association with AF. The SNPs in this locus most strongly associated with AF are located in an intergenic region that is 78 to 157 kb distal to the closest gene PITX2. PITX2 is a candidate gene for AF, due to its role in left/right patterning during heart development and expression found only in the left vs. right atrium. Our lab performed RNA-seq on human atria and identified a long intergenic non-coding RNA (linc-RNA) adjacent to PITX2 that is expressed with the same left/right atrial asymmetry as PITX2. We hypothesized that there may be functional genetic variants in the 4q25 region that directly affect expression of PITX2 or the linc-RNA adjacent to PITX2. Further, we hypothesized that the linc-RNA may control in trans the expression of genes that can alter cellular electrophysiology and lead to AF susceptibility. First we used qPCR to determine whether the expression of PITX2 and this linc-RNA were correlated in 199 human left atria samples. We found the expression of these genes to be highly and positively correlated with an r2 value of 0.47 (p < .0001). We obtained genome-wide SNP information for each of these subjects using an Illumina SNP microarray. We determined the association of SNPs on chromosome 4 with the expression levels of PITX2 and the linc-RNA by performing expression QTL (eQTL) analysis. In the 4q25 region, common genetic variation was more strongly associated with expression of the linc-RNA than PITX2. Two of the strongest linc-RNA eQTL SNPs (p-value ~10-4) were associated with AF with a p-value of ~10 15. We are now starting functional studies to determine if this linc-RNA can modulate gene expression in trans by transfection of a linc-RNA expression plasmid into HEK-293 cells. We plan to determine the effects of over expression this linc-RNA on global gene expression profiles using expression arrays and RNA-seq
Abstract 111: High Activity Gating of Caveolae-Targeted L-Type Ca
2+
Channels Can Initiate Pathological Hypertrophy
Authors: Makarewich Catherine A , Correll Robert N , Zhang Hongyu , Gao Hui , Yang Baohua , Berretta Remus M , Rizzo Victor , Molkentin Jeffery D , Houser Steven R
Published: July 2021

Introduction:
The source of Ca
2+
that activates pathological cardiac hypertrophy is not clearly defined. We hypothesize that high activity gating of L-type Ca
2+
channels (LTCCs) localized in caveolae signaling microdomains stabilized by caveolin-3 (Cav-3) provides the Ca
2+
influx that locally activates calcineurin (Cn)-mediated nuclear factor of activated T-cells (NFAT) to induce hypertrophic gene expression. We generated novel reagents that specifically activate or inhibit the gating of LTCCs in caveolae for analysis of the hypertrophic program, as well as any potential effect on ICa and contraction.

Methods and Results:
We targeted the known LTCC inhibitory protein Rem or an LTCC subunit (β2a) that promotes high activity gating specifically to caveolae by fusing them to a caveolin-binding domain peptide (termed Cav-Rem and Cav-β2a). We infected adult feline left ventricular myocytes (AFLVMs) with adenoviruses containing Cav-Rem or Cav-β2a for membrane localization and functional studies. NFAT nuclear translocation was determined by co-infecting AFLVMs with ad-NFAT-GFP and either ad-Cav-Rem or ad-Cav-β2a and pacing cells to induce Ca2+ influx mediated nuclear NFAT-GFP translocation. Membrane fractionation experiments showed that Cav-3 membrane domains contain 26.2 +/- 12.7% of membrane targeted LTCCs and blocking these with Cav-Rem eliminated a small fraction of the LTCC current (<15%) and almost all Ca2+ influx induced NFAT nuclear translocation (>90%), but did not reduce myocyte contractility. Conversely, selective enhancement of LTCC activity within caveolae with Cav-β2a caused a significant increase in NFAT nuclear translocation (>70%) but had no significant effect on contractility.

Conclusions:
We provide proof of concept that specific Cav-targeted reagents can be used to enhance or inhibit LTCC activity within caveolae microdomains to amplify or block the hypertrophic response. Our results suggest that high activity gating of LTCCs, which is known to be present in the hypertrophied failing human heart, can activate signaling pathways linked to cardiac hypertrophy. Selectively inhibiting these caveolae localized LTCCs could be a novel mechanism to block pathological hypertrophy without reducing cardiac contractility.
Abstract 122: Cardiac mTOR Preserves Cardiac Function Following Ex Vivo Ischemia-Reperfusion in Diet-Induced Obese Mice
Authors: Aoyagi Toshinori , Matsui Takashi
Published: July 2021

The risk of heart failure following myocardial infarction is higher in diabetic patients than nondiabetic patients. The mammalian target of rapamycin (mTOR), a key downstream molecule of insulin-phosphoinositide 3-kinase (PI3K)-Akt signaling pathway, plays an important role in cardioprotection. However, the role of cardiac mTOR in ischemic injury in metabolic syndrome has not been well defined. To address this question, we studied the effect of overexpressing cardiac mTOR on cardiac function following ischemia/reperfusion (I/R) in mice with high-fat diet (HFD)-induced obesity. In this study, we used transgenic mice with cardiac-specific overexpression of mTOR (mTOR-Tg) as reported previously. mTOR-Tg and WT mice at 6 weeks old were fed HFD (60% fat by calories) ad libitum for 14 weeks. Control mTOR-Tg and WT mice were fed a normal chow diet (NCD). At 14 weeks after HFD, glucose and insulin tolerance tests demonstrated that HFD generated glucose intolerance and insulin resistance in both mTOR-Tg (n=20) and WT (n=24) mice. Body weight (BW) and heart weight (HW) were significantly higher in HFD mice than SCD mice (p<0.001 for BW in both strains; p<0.001 and p<0.01 for HW/tibia length, WT and mTOR-Tg, respectively) but there was no difference in BW or HW between HFD-mTOR-Tg and HFD-WT mice. Hearts from all four groups were subjected to global I/R (20 min ischemia, 40 min reperfusion) in the ex vivo Langendorff perfusion model. Baseline left ventricular developed pressure (LVDP) was higher in HFD mice than NCD mice in both strains [185.8 ± 10.7 vs. 143.6 ± 5.0 mmHg, HFD-WT (n=11) vs. NCD-WT (n=10) mice, p<0.01; 178.6 ± 10.1 vs. 135.0 ± 6.3, HFD-mTOR-Tg (n=8) vs. NCD-mTOR-Tg (n=11) mice, p<0.01]. Functional recovery after I/R was significantly lower in HFD-WT mice than NCD-WT mice (percent recovery of LVDP, 15.3 ± 5.4 vs. 44.6 ± 6.3 %, HFD-WT vs. NCD-WT mice, p<0.01). Intriguingly, there was no significant difference in LVDP recovery between HFD-mTOR-Tg and NCD-mTOR-Tg mice (36.5±10.8 vs. 58.8±6.0 %, HFD-mTOR-Tg vs. NCD-mTOR-Tg mice, n.s.). These findings suggest that cardiac mTOR is sufficient to substantially limit the metabolic syndrome-induced cardiac dysfunction following I/R in a mouse model of obesity with glucose intolerance and insulin resistance.
Abstract 129: Suppressor of Cytokine Signaling 1 in Dendritic Cells Inhibits Myocardial Inflammation in Experimental Autoimmune Myocarditis
Authors: Tajiri Kazuko , Imanaka-Yoshida Kyoko , Hiroe Michiaki , Shimojo Nobutake , Sakai Satoshi , Aonuma Kazutaka , Yasutomi Yasuhiro
Published: July 2021

Introduction:
Autoimmunity is considered to play an important role in the development of myocarditis and dilated cardiomyopathy. Recent reports have indicated that a subgroup of myocarditis patients may benefit from immune-targeted therapies. Suppressor of cytokine signaling1 (SOCS1) is an intracellular, cytokine-inducible protein that regulates the responses of immune cells to cytokines. We therefore hypothesized that overexpression of SOCS1 may inhibit the inflammation of myocarditis and cardiomyopathy.

Methods and Results:
Myocarditis was induced by subcutaneous immunization with cardiac specific peptides derived from α-myosin heavy chain in BALB/c mice on days 0 and 7. Plasmid DNA encoding SOCS1 (pSOCS1) was injected intraperitoneally into mice on days 0, 5 and 10. pSOCS1 treatment significantly decreased heart-to-body weight ratios and the number of infiltrating cells in the heart. Echocardiography showed preserved contractile function in pSOCS1-treated mice. Although autoimmune myocarditis is a CD4+ T cell-mediated disease, pSOCS1 treatment does not have a direct suppressive effect on autoreactive T-cell activation. The introduced pSOCS1 suppressed proinflammatory cytokine production and STAT1 phosphorylation in dendritic cells (DCs). In addition, the proliferative responses of autoreactive CD4+ T cells co-cultured with DCs from pSOCS1-treated mice were much weaker than those of cells cultured with DCs from control plasmid-injected mice. These results suggested that the inoculated pSOCS1 may have been transfected into DCs and impaired DC function in vivo.

Conclusion:
The administration of pSOCS1 protected mice from the development of experimental autoimmune myocarditis, which was mediated by the inhibition of DC function that in turn reduced the activation of autoreactive CD4+ T cells.
Abstract 137: Deficiency of Senescence Marker Protein 30 Exacerbates Doxorubicin-Induced Cardiac Dysfunction in Mice
Authors: Miyata Makiko , Suzuki Satoshi , Misaka Tomofumi , Saitoh Shu-ichi , Takeishi Yasuchika
Published: July 2021

Background:
Senescence marker protein 30 (SMP30) was originally identified as an aging marker protein in the rat liver. The expression of SMP30 decreases with aging androgen-independently. Doxorubicin (DOX) has been used as a potent anticancer agent, but serious cardiotoxicity precludes its use in a wide range of patients. SMP30 may have anti-oxidative and anti-apoptosis functions in several organs, but functional role of SMP30 has not been rigorously examined in the heart. We hypothesized that SMP30 has cardio-protective function by anti-aging and anti-oxidant effects on DOX-induced cardiac dysfunction.

Method and Results:
Male SMP30 knockout (SMP30KO) and age-matched littermate male wild-type (WT) mice at 12-14 weeks of age were given intraperitoneal injections of DOX (20 mg/kg) or saline. Seven days after DOX injection, echocardiography revealed that left ventricular ejection fraction in DOX-treated SMP30KO mice was more severely reduced than in DOX-treated WT mice (40.9 ± 3.1% vs. 46.9 ± 4.9%, P<0.01). Morphological examination of myocardial sections showed fibrotic change in DOX-treated SMP30KO mice significantly increased compared to DOX-treated WT mice (3.2 ± 0.5% vs. 1.3 ± 0.2%, P<0.01). Generation of reactive oxygen species assessed by dihydroethidium staining was greater in DOX-treated SMP30KO mice than DOX-treated WT mice (166.9 ± 8.6% vs. 131.6 ± 5.8%, P<0.01). Moreover, apoptotic signaling pathways such as caspase-3 activity (1.8 ± 0.1% vs. 1.1 ± 0.2%, P<0.01), bax/bcl-2 ratio (2.4 ± 0.3% vs.1.6 ± 0.2%, P<0.05) and phosphorylation activity of c-Jun N-terminal kinase (1.6 ± 0.3 vs. 1.0 ± 0.1, P<0.05) were significantly elevated in the SMP30KO mice compared with WT mice after DOX injection. The numbers of TUNEL-positive nuclei in the myocardium were higher in DOX-treated SMPKO mice than in DOX-treated WT mice (0.15 ± 0.02% vs. 0.08 ± 0.01%, P<0.01).

Conclusions:
The results of this study demonstrated that DOX-induced cardiotoxicity is aggravated in SMP30KO mice by exacerbating of superoxide generation, leading to enhanced apoptosis of cardiomyocytes. SMP30 has a cardio-protective role by anti-apoptotic and anti-oxidative effects in DOX-induced cardiotoxicity.
Abstract 144: Interstitial Formation of Adenosine and Inosine Is Proarrhythmic Through Adenosine A1 Receptor in the Developing Heart
Authors: Robin Elodie , Sabourin Jessica , Raddatz Eric
Published: July 2021

We previously established that exogenous adenosine (ADO) induces transient arrhythmias in the developing heart via the ADO A1 receptor (A1AR) and downstream activation of NADPH oxidase/ERK and PLC/PKC pathways.

We assessed the hypothesis that both endogenous ADO and its derived compound inosine (INO) can simultaneously act in an autocrine/paracrine manner and induce rhythm and conduction disturbances.

The validated model of the spontaneously beating heart obtained from 4-day-old chick embryos was used. The basal ADO/INO ratio determined by HPLC was approximately 10. Blockade of Equilibrative Nucleoside Transporters (ENTs) by dipyridamole, which is known to increase interstitial ADO level, induced transient atrial dysrhythmias and atrio-ventricular blocks in 70% of the hearts (n=30) and, in parallel, increased ERK2 phosphorylation. When extracellular conversion of AMP to ADO by ecto-5’-nucleotidase/CD73 was inhibited by AOPCP, the dipyridamole-induced arrhythmias were prevented. The dipyridamole-induced arrhythmias and ERK2 phosphorylation were prevented by the specific antagonist of the A1AR (DPCPX) but not by antagonists of A2AAR (
SCH58261
), A2BAR (MRS1754) or A3AR (MRS1523). Interestingly, inhibition by EHNA of the conversion of ADO to INO by adenosine deaminase was not proarrhythmic whereas exogenous INO (500 µM) induced arrhythmias which were prevented by DPCPX.

In conclusion, these findings suggest that endogenous ADO and INO in the interstitial compartment of the developing heart can provoke transient pacemaking and conduction disturbances via A1AR and downstream ERK activation in an autocrine/paracrine manner. Overall, our results provide new insights into the mechanisms underlying cardiac dysfunction when a hypoxic-ischemic episode induces local overproduction of ADO and INO
Abstract 174: Late I
Na
Inhibition with Ranolazine Blunts Doxorubicin-Induced Cardiac Dysfunction and Remodeling in Mice
Authors: Tocchetti Carlo G , Coppola Carmela , Rea Domenica , Quintavalle Cristina , Barbieri Antonio , Piscopo Giovanna , Palma Giuseppe , Gala Marianna , Luciano Antonio , Giudice Aldo , Iaffaioli Rosario V , Scala Stefania , Condorelli Gerolama , Arra Claudio , Maurea Nicola
Published: July 2021

Background.
Doxorubicin (DOX) produces a well-known cardiomyopathy through multiple mechanisms, which include, among many, Ca2+ overload due to reduced SERCA2a activity and inappropriate opening of the RyR2, and impaired myocardial energetics. DOX generate Reactive Oxygen and Nitrogen Species (ROS and RNS), posing the heart at increased demand for oxygen, setting the stage for metabolic ischemia that also activates late I
Na
, target of ranolazine (RAN). Here, we aim at assessing whether RAN, diminishing intracellular Ca2+ through inhibition of late I
Na
, and enhancing myocardial glucose utilization (and/or reverting impairment of glucose utilization caused by chemotherapy) blunts DOX cardiotoxicity.

Methods.
We measured left ventricular (LV) end-diastolic and end-systolic diameters (LVEDD and LVESD), and evaluated LV function with fractional shortening (FS) by echocardiography in C57BL6 mice, 2-4 mo old, pretreated with RAN (370mg/kg/day, a dose comparable to the one used in humans) per os for 3 days. RAN was then administered for additional 7 days, alone and together with DOX (2.17mg/kg/day ip), according to our well established protocol. Hearts were then excised, mRNA expression was analyzed by RT PCR.

Results
. After 7 days with DOX, hearts were dilated with depressed function: LVEDD and LVESD increased from 2.8±.03 and 1.1±.03mm to 3.04±.06 and to 1.55±.08mm, respectively (both p<.005 vs sham). FS decreased to 50±2%, p=.002 vs 60±1% (sham). RAN alone did not change LV dimensions nor function. Interestingly, mice treated with RAN and DOX had smaller LVEDD and LVESD (2.85±.05 and 1.22±.03mm, respectively, both p<.05 vs DOX alone), with a better cardiac function: FS was 57±1%, p=.01 vs DOX alone. DOX-cardiotoxicity was accompanied by significant elevation in CTGF and MMP2 genes, showing alterations in extracellular matrix remodeling. Cardiac function improvement with RAN was paralleled by significantly lower levels of these same genes compared to DOX alone.

Conclusions
. RAN blunts DOX cardiotoxic effects in mice, improving remodeling and function. We plan to test RAN as a cardioprotective agent with other antineoplastic cardiotoxic drugs in mice, and to better characterize the cardioprotective mechanisms of RAN in all these settings.
Abstract 177: Overexpression of Cardiac Troponin I-Interacting Kinase, a Cardiac-Specific MAPKKK, Promotes Cardiac Dysfunction
Authors: Tang Hao , Xiao Kunhong , Mao Lan , Rockman Howard A , Marchuk Douglas A
Published: July 2021

Cardiac Troponin I-interacting kinase (TNNI3K) is a cardiac specific kinase whose biological function remains largely unknown. We have recently shown that TNNI3K expression greatly accelerates cardiac dysfunction in mouse models of cardiomyopathy, indicating an important role in modulating disease progression. To further investigate TNNI3K kinase activity in vivo, we have generated transgenic mice expressing both wild-type and kinase-dead versions of the human TNNI3K protein. Importantly, we show that the increased TNNI3K kinase activity induces mouse cardiac hypertrophy, and the kinase activity is required to accelerate disease progression in a left-ventricular pressure overload model of mouse cardiomyopathy. We demonstrate the clinical relevance of these observations by identifying two potential missense mutations near the kinase activation loop of TNNI3K in idiopathic dilated cardiomyopathy (DCM) human patients. Using an in vitro kinase assay and proteomics analysis, we show that TNNI3K is a dual-function kinase with Tyr and Ser/Thr kinase activity. Using antisera to TNNI3K, we show that TNNI3K protein is located at the sarcomere Z disc. These combined data suggest that TNNI3K mediates cell signaling to modulate cardiac response to stress. The essential role of the kinase activity makes TNNI3K a strong potential pharmaceutical target of kinase inhibitors for heart disease.
Abstract 182: Class I Histone Deacetylase Inhibition Represses the Upregulation of Sodium-Calcium Exchanger Expression in Cardiac Hypertrophy
Authors: Mani Santhosh K , Chernysh Olga , Li Mona S , Renaud Ludivine , Janech Michael G , Menick Donald R
Published: July 2021

Background:
Histone deacetylases (HDACs) play an important role in the alteration of gene expression during cardiac hypertrophy and failure. Our previous study demonstrated that acetylated Nkx2.5 is associated with the Class I/II HDAC complex, HDAC5/1/2 at the Ncx1 promoter, and deacetylated Nkx2.5 is associated with the transcriptional activator and histone acetylase, p300 in a mutually exclusive manner. Inhibition of HDACs by the Class I/II inhibitor, trichostatin A (TSA) prevents deacetylation of Nkx2.5 and recruitment of p300 to the Ncx1 promoter, thereby repressing its upregulation.

Objective:
To assess the specific roles of HDAC1, 2 and 5 in the regulation of Ncx1 gene expression and determine the specific Nkx2.5 lysine(s) undergoing acetylation or deacetylation, which mediates Ncx1 upregulation.

Results:
Treatment of isolated adult cardiomyocytes with the selective class I HDAC inhibitor, BML210, prevented alpha- and beta-adrenergic stimulated upregulation of Ncx1 expression, whereas treatment with the specific class II HDAC inhibitor, dPAHA did not. Interestingly, the HDAC5 knockout prevented NCX1 upregulation after 72 hr trans-aortic constriction (TAC) in mice. In order to determine which Nkx2.5 lysine(s) is deacetylated by HDAC1/2, we performed mass spectrometry analysis. The Nkx2.5 gene contains 15 lysine moieties, and LC-MS/MS analysis demonstrates that Nkx2.5 is acetylated on two conserved lysine residues.

Conclusion:
Class I HDAC activity is required for Ncx1 expression but not class II HDAC activity (HDAC5). However, the loss of HDAC5 prevents Ncx1 upregulation because it may act as a scaffold to recruit the factors required for Ncx1 promoter activation. These results suggest that HDAC inhibition may represent a novel therapeutic modality for hypertrophy and heart failure.
Abstract 187: Overexpression of Calpastatin Enhances Cardiotoxicity Induced by Doxorubicin
Authors: Peng Tianqing , Wang Yanpeng , Ma Jian
Published: July 2021

Background:

Doxorubicin causes damage to the heart, which may present as cardiomyopathy. However, the mechanisms by which doxorubicin induces cardiotoxicity remain not fully understood and no effective prevention for doxorubicin cardiomyopathy is available. Calpains, a family of calcium-dependent thiol-proteases, have been implicated in cardiovascular diseases. Their activities are tightly controlled by calpastatin. This study employed transgenic mice over-expressing calpastatin to investigate the role of calpain in doxorubicin-induced cardiotoxicity.

Methods and Results:

Doxorubicin treatment decreased calpain activities in cultured neonatal mouse cardiomyocytes and
in vivo
mouse hearts. Over-expression of calpastatin or incubation with pharmacological calpain inhibitors enhanced caspase-3 activity and DNA fragmentation in both neonatal and adult cardiomyocytes induced by doxorubicin. Inhibition of calpain also induced down-regulation of phosphorylated protein kinase B (AKT, Thr308), and a concomitant reduction in glycogen synthase kinase-3beta (GSK-3β) phosphorylation (Ser9) in doxorubicin-treated cardiomyocytes. Blocking AKT further increased doxorubicin-induced cardiac injuries, suggesting the effects of calpain inhibition may be mediated by inactivating the AKT/GSK-3β signaling. In an
in vivo
model of doxorubicin-induced cardiotoxicity, over-expression of calpastatin decreased calpain activities and exacerbated myocardial dysfunction as assessed by echocardiography and hemodynamic measurement in transgenic mice (C57BL/6) 5 days after doxorubicin injection. The five-day mortality was higher in transgenic mice (29.16%) compared with their wild-type littermates (8%) after doxorubicin treatment.

Conclusions:

Over-expression of calpastatin enhances doxorubicin-induced cardiac injuries through inhibiting calpain and compromising AKT survival signaling. Thus, calpains may protect cardiomyocytes against doxorubicin-induced cardiotoxicity.
Abstract 189: Activation of Voltage-Dependent Anion Channel 2 Suppresses Ca
2+
-Induced Cardiac Arrhythmia
Authors: Chen Jaunian , Schredelseker Johann , Shimizu Hirohito , Huang Jie , Lu Kui , Franklin Sarah , Vondriska Thomas , Goldhaber Joshua I , Kwon Ohyun
Published: July 2021

Abnormal Ca2+ handling in cardiac muscle cells is associated with a wide range of human cardiac diseases, including heart failure and cardiac arrhythmias. Zebrafish tremblor (tre) mutant embryos manifest unsynchronized cardiac contractions due to a Ca2+ extrusion defect in cardiomyocytes and thus are used as an animal model for aberrant Ca2+ homeostasis-induced cardiac arrhythmia. To further dissect molecular mechanisms regulating cardiac Ca2+ homeostasis, we conducted a chemical suppressor screen on tre and found that efsevin, a synthetic compound, potently suppresses cardiac fibrillation and restores rhythmic cardiac contractions in tre embryos. In addition, the treatment with efsevin blocks the propagation of arrhythmogenic Ca2+ waves and accelerates the decay phase of Ca2+ sparks in adult murine cardiomyocytes under Ca2+ overload conditions, demonstrating that efsevin modulates Ca2+ handling in both embryonic and adult cardiac tissues. Through a biochemical pulldown assay, we identified a direct interaction between efsevin and VDAC2, a mitochondrial outer membrane voltage dependent anion channel. Overexpression of VDAC2 restores synchronized cardiac contraction in tre and knocking down VDAC2 activity abolishes the rescue effect of efsevin on tre, suggesting that efsevin modulates cardiac Ca2+ homeostasis by potentiating VDAC2 activity. We further showed that enhancing mitochondria Ca2+ uptake by overexpressing MICU or MCU suppresses cardiac fibrillation in tre just like VDAC2 does. Interestingly, this suppressive effect is absent in tre/vdac2 double deficient embryos and co-expression of VDAC2 and MICU or MCU results in synergistic rescue effect on tre, indicating a critical role for mitochondria in regulating cardiac Ca2+ handling and rhythmicity and suggesting that VDAC2 functions as a gate for transporting Ca2+ across the outer membrane. Taken together, our findings identify efsevin as a potent pharmacological tool to modulate cardiac Ca2+ handling, suggest a critical role of mitochondria in the control of cardiac rhythmicity and establish VDAC2 as a modulator of cardiac Ca2+ handling and a potential therapeutic target for the treatment of arrhythmias.
Abstract 207: Differential Regulation of Ubiquitin-Proteasomal System in Right Ventricular Versus Left Ventricular Hypertrophy and Failure
Authors: Rajagopalan Viswanathan , Zhao Mingming , Reddy Sushma , Fajardo Giovanni , Dewey Shannamar , Gomes Aldrin , Bernstein Daniel
Published: July 2021

Background:

While the role of Ubiquitin-Proteasomal System (UPS) in left ventricular hypertrophy (LVH) and failure (LVF) has been controversial, the role of UPS in the right, RVH/RVF is unknown. Given the greater percent increase in RV mass and protein with RV vs. LV afterload stress, we hypothesized that UPS alterations could play an important role in RVH and RVF.

Methods:

Using our pulmonary artery constriction (PAC) model to induce RVH/RVF by 8d (n=13-14), UPS gene and protein expression, ubiquitination, oxidation (carbonylation), and enzyme activity were measured in PAC vs. sham controls. Proteasomal inhibitors epoxomicin (0.5 mg/kg/d) or MG132 (10 µg/kg/d) were administered from 1d before to 7d after surgery (n=3-6).

Results:

RVW/BW was increased by 109.3% in PAC mice. All mice developed progressive RV dilation, septal shift and decrease in RV function. UPS function, measured as 26S ß
5
chymotrypsin-like activity (71.3±3.9 vs. 100±8.3%; p<0.05) and 20S ß
1
caspase-like activity (66.9±3.2 vs. 100±6.9%; p<0.001), was decreased. 20S protein expression was unchanged whereas the 19S regulatory subunit Rpt5 was increased (3.3±0.5 vs. 1.4±0.2; p<0.05). UCHL1 deubiquitinase (0.3±0.04 vs. 0.05±0.004; p<0.0001) and Smurf1 E3 ubiquitin ligase (3369±538 vs. 1249±75.6; p<0.01) were increased and both poly-ubiquitinated proteins (877600±50550 vs. 639700±45050; p<0.05) and free-ubiquitins (373102±65859 vs. 177318±24188) were also increased. Carbonylation was unchanged. Pro-apoptotic Bax was increased (0.06±0.004 vs. 0.02±0.001; p<0.0001), and anti-apoptotic Bcl-2 decreased (0.2±0.03 vs. 0.5±0.08; p<0.05) with a 6-fold increase in Bax/Bcl-2 ratio. Proteasomal inhibition further increased free ubiquitin levels 2.6-fold (p<0.0001). However, neither the degree of RVH, nor decrease in RV function was altered.

Conclusion:

UPS has been shown to be both activated/inhibited and cardioprotective/cardiotoxic in LVH/LVF. In RVH/RVF, despite a doubling in RV mass, UPS activity is decreased, associated with increased ubiquitinated proteins and Bax/Bcl-2 ratio. Further proteasomal inhibition does not hasten RVF, suggesting that the failing RV is resistant to further proteasomal inhibition, or a limited role for the UPS in the progression from RVH to RVF.
Abstract 211: Aldehyde Dehydrogenase 2 Deficiency Aggravates Cardiac Dysfunction Elicited by Endoplasmic Reticulum Stress Induction
Authors: Sun Aijun , Liao Jianquan , Qing Xieye , Jin Xueting , Isse Toyoshi , Kawamoto Toshihiro , Zou Yunzeng , Ge Junbo
Published: July 2021

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) has been characterized as an important mediator of endogenous cytoprotection in the heart. This study was designed to examine the role of aldehyde dehydrogenase-2 knockout (KO) in the pathogenesis of heart underwent endoplasmic reticulum (ER) stress induction. Wild-type (WT) and ALDH2 KO mice were subjected to tunicamycin challenge and echocardiographic examination was performed. Protein levels of GRP78, p-eIF2α, CHOP, phosphorylation of Akt, p47phox NADPH oxidase, and 4-hydroxynonenal were determined by Western blot analysis. Cytotoxicity and apoptosis were estimated by MTT assay and caspase-3 activity respectively. ALDH2 deficiency exacerbated cardiac dysfunction and increased the protein levels of ER stress markers after ER stress induction characterized by the changes of ejection fraction and fractional shorting, when compared with WT mice. In vitro, tunicamycin significantly increased in the levels of GRP78, p-eIF2, CHOP and p47phox NADPH oxidase, which was exacerbated by ALDH2 knockdown and abolished by ALDH2 overexpression. Overexpression of ALDH2 abrogated tunicamycin-induced dephosphorylation of Akt. Inhibition of PI3-K with LY294002 did not negatively affect the inhibition of ER stress markers conferred by ALDH2, but reversed the anti-apoptotic role of ALDH2, which may be associated with p47phox NADPH oxidase. These results suggest that ALDH2 was implicated in the regulation of ER stress and ER stress-induced apoptosis. The protective role of ALDH2 against cell death induced by ER stress was probably mediated by Akt signaling via p47phox NADPH oxidase. These findings indicate a critical role of ALDH2 in the pathogenesis of ER stress in heart disease.
Abstract 212: Hydrogen Peroxide Acutely Stimulates Cardiogenic Gene Expression in Cardiac Progenitor Cells
Authors: Pendergrass Karl D , Davis Michael E
Published: July 2021

Following acute myocardial infarction, billions of myocytes are lost to cell death. The damage is regional and lost cells are replaced with a collagen scar. One potential therapy to delay or prevent progression into heart failure is regeneration of the damaged myocardium through cell therapy with cardiac progenitor cells (CPC). Reactive oxygen species, specifically hydrogen peroxide, elicit varying responses from different stem/progenitor cells. In the present studies, we sought to determine the effect of acute H2O2 treatment on CPC survival and differentiation. CPCs were isolated and cultured with leukemia inhibitory factor (LIF) to retain their stem-like qualities. CPCs were allowed to differentiate in the absence of LIF for up to 5 days + H2O2. H2O2 (100μM) significantly increased expression of the smooth muscle marker, alpha smooth muscle actin (αSM) by Day 2 as compared to time-matched controls (Ctl: 1.8+1.3 vs 100 μM: 8.5+1.1; p<0.001; N=3). We also observed a trend for an increase in smooth muscle 22 alpha (SM 22α) gene expression by Day 2. Interestingly, by Day 5 the stimulatory effect of 100 μM H2O2 treatment on α SM and SM 22α was reversed and significantly decreased compared to Day 2 (D5: α SM: 0.43+0.05, SM 22α: 0.41+0.2 vs. D2: SM 22α: 10.58+2.3; p<0.01; N=3-4). Evaluation of the endothelial marker VEGFR-2 (Flk-1) showed a trend for an increase in gene expression by Day 2 following 100 μM H2O2 treatment compared to the time-matched control. We also observed an anti-apoptotic effect on CPCs following serum removal, in which 2 days of 100μM H2O2 pretreatment lead to approximately a 55% decrease in cell death compared to untreated CPCs (Ctl: 19.1+2.4 vs 100 μM: 10.5+1.7; p<0.05; N=3-4). The protective effect of the H2O2 pretreatment could be attributed to an increase in anti-oxidative enzymatic capacity in CPCs. There was a trend for an increase in catalase gene expression. In conclusion, our results showed that acute H2O2 preconditioning exerted a stimulatory effect on smooth muscle gene expression and an anti-apoptotic effect compared to time-matched Control CPCs. Furthermore, acute H2O2 preconditioning may aid in directing CPC differentiation towards a vascular phenotype and angiogenesis in the infarcted myocardium, which may prevent or delay heart failure.
Abstract 219: Cardiac Angiogenic Imbalance Leads to Peripartum Cardiomyopathy
Authors: Arany Zolt , Patten Ian , Rana Sarosh , Shahul Sajid , Rowe Glenn , Jang Cholsoon , Liu Laura , Hacker Michele , Rhee Julie , Mitchell John , Mahmood Feroze , Hess Phil , Farrell Caitlin , Koulisis Nicole , Khankin Eliyahu , Burke Suzanne , Tudorache Igor , Bauersachs Johann , del Monte Federica , Hilfiker-Kleiner Denise , Karumanchi Ananth
Published: July 2021

Peri-partum cardiomyopathy (PPCM) is a frequently fatal disease that affects women near delivery, and occurs more frequently in women with pre-eclampsia and/or multiple gestation. The etiology of PPCM, or why it associates with pre-eclampsia, remains unknown. We show here that PPCM is associated with a systemic angiogenic imbalance, accentuated by pre-eclampsia. Mice that lack cardiac PGC-1α, a powerful regulator of angiogenesis, develop profound PPCM. Importantly, the PPCM is entirely rescued by pro-angiogenic therapies. In humans, the placenta in late gestation secretes VEGF inhibitors like soluble Flt1 (sFlt1), and this is accentuated by multiple gestation and pre-eclampsia. This anti-angiogenic environment is accompanied by sub-clinical cardiac dysfunction, the extent of which correlates with circulating levels of sFlt1. Exogenous sFlt1 alone caused diastolic dysfunction in wildtype mice, and profound systolic dysfunction in mice lacking cardiac PGC-1α. Finally, plasma samples from women with PPCM contained abnormally high levels of sFlt1. These data strongly suggest that PPCM is in large part a vascular disease, caused by excess anti-angiogenic signaling in the peri-partum period. The data also explain how late pregnancy poses a threat to cardiac homeostasis, and why pre-eclampsia and multiple gestation are important risk factors for the development of PPCM.