bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 221: Prognostic Role of elevated Myeloperoxidase in Patients with Acute Coronary Syndrome: A Systemic Review and Meta-Analysis.
Authors: Kolodziej Andrew R , Campbell Charles L , Charnigo Richard , Twerenbold Raphael , Mueller Christian , Ziada Khaled M , Abdel-Latif Ahmed
Published: July 2021

BACKGROUND:
It has been shown that Myeloperoxidase (MPO) is intimately involved in pathogenesis of atherosclerosis and Acute Coronary Syndrome (ACS). Small studies have shown that high levels of MPO are a poor prognostic factor in patients presenting with ACS. However, due to the small nature of these studies, the relationship between MPO and outcomes has not been confirmed. Here we aimed to examine the prognostic value of MPO in patients with ACS.

METHODS:
We performed a meta-analysis to compare the long-term prognosis of ACS patients with high MPO and low MPO levels. The literature was retrieved by formal searches of electronic databases (PubMed, EMBASE, Medline, OVID, and web of knowledge) from inception to November 2013. A total of 16 trials were included in this meta-analysis involving 10572 patients. Data were analyzed using random-effects model and study quality was assessed using appropriate scales.

RESULTS:
High MPO group was associated with overall worse outcomes than low MPO group in terms of recurrent myocardial infarction (9% [211 of 2336] vs. 7.7% [240 of 3101], odds ratio [OR] 1.4, 95% confidence interval [CI]: 0.92-2.15, p 0.11); all cause mortality (8% [236 of 2920] vs. 5% [209 of 4263], OR 1.83, 95% CI: 1.31-2.54, p <0.0004) and Major Adverse Cardiovascular Events (MACE) (24% [334 of 1400] vs. 14% [133 of 949], OR 2.04, 95% CI: 1.46-2.85, p< 0.0001) (Figure).

CONCLUSIONS:
In this meta-analysis examining the long-term outcomes in ACS patients, high MPO levels were associated with worse outcomes. These observations support prospective trials tailoring more aggressive therapy to patients with suspected worse prognosis.
Abstract 224: 4-phenylbutyric Protects The Heart From Heart Failure, Cardiac Rupture And Remodeling By Alleviating Cardiac Apoptosis And Fibrosis In A Mouse Model Of Myocardial Infaction
Authors: Luo Tao , Chen Baihe , Wang Xianbao , Wang Yanping , Kitakaze Masafumi , Liao Yulin , Yan Liang
Published: July 2021

Evidences showed that endoplasmic reticulum (ER) stress has involved in cardiac hypertrophy and inhibition of ER stress exerts an effective cardioprotective effect. But little is known about the effect of ER stress inhibition on myocardial infarction (MI). The aim of this study is to investigate whether direct inhibition of ER stress by 4-phenylbutyric acid (4-PBA), an ER stress chemical chaperone, could attenuate MI induced by left coronary artery (LCA) ligation in male C57BL/6 mice. Either NaCl or 4-PBA (20 mg/kg/d) alone was intra-peritoneal injected to the mice immediately after MI for 4 weeks. Heart failure, cardiac rupture and remodeling occurred after MI. There was 52.5%
vs
24% death rate in control group
vs
4-PBA group during the early phase after MI (2-7 days). The survivals underwent cardiac remodeling with impaired systolic and diastolic functions at the end. The ventricular aneurysm and fibrosis were much smaller in 4-PBA group and the heart functions were improved. Western blot showed that the ER stress markers, Bax, Caspase 3, TGFβ1 and Smad 2/3 in the heart tissues of the control group were significantly increased after MI, in which Bax and Caspase 3 were increased markedly in the early phase of MI, while TGFβ1 and Smad 2/3 were increased obviously in the late phase. 4-PBA decreased those protein levels in the early and late phases of MI respectively. These findings indicate that inhibition of ER stress using 4-PBA could be an effective therapeutic agent protects the heart from post-infarcted heart failure, cardiac rupture and remodeling.
Abstract 236: Sirt6 Heterozygosity Exacerbates Atherosclerosis in Apolipoprotein E Deficient Mice
Authors: Liu De-Pei , Zhang Zhu-Qin , Ren Si-Cong , Li Zuo-Zhi , Tan Ying , Tang Xiao-Qiang , Hao De-Long , Guo Zhi-Chen , Zhao Xiang , Chen Hou-Zao
Published: July 2021

Sirt6 is a member of the class III histone deacetylase family and is reported to promote longevity. Whether Sirt6 is involved in atherosclerosis, one aging associated disease and the major cause of cardiovascular diseases, is unknown. We investigated effects of Sirt6 on atherosclerosis development. We found that in human atherosclerotic plaques, Sirt6 expression was decreased. Sirt6+/-ApoE-/- mice exhibited increased atherosclerosis development and decreased plaque stability than ApoE-/- mice. We found that Sirt6 downregulation showed increased expression of NKG2D ligands (H60b in mice and MICA/B in human). Sirt6 bound to promoters of these genes and regulated the H3K9 acetylation levels. Thus, atherosclerosis development was promoted by Sirt6 heterozygosity and epigenetic modification of NKG2D ligand expression is involved in this process.
Abstract 238: miR-222 is Necessary for Exercise-induced Cardiac Growth and Protects Against Pathological Cardiac Remodeling
Authors: Liu Xiaojun , Xiao Junjie , Zhu Han , Wei Xin , Platt Colin , Damilano Federico , Xiao Chunyang , Bezzerides Vassilios , Rosenzweig Anthony
Published: July 2021

Exercise induces cardiac growth, protects against adverse remodeling, and may also induce a regenerative response. Since microRNAs (miRNA) play important roles in cardiovascular disease, we investigated their role in the cardiac exercise response.

We used the TaqMan rodent miRNA array to profile cardiac miRNA expressed at three weeks in two exercise models (swimming, running) compared to sedentary controls. Sixteen concordantly regulated miRNAs were identified and validated in both models, and examined for functional effects in neonatal rat ventricular cardiomyocytes (CMs). miR-222 was upregulated ~two-fold in both models and increased CM size (22%, p<0.01) and proliferation markers (EdU and Ki67, p<0.01). Bioinformatic and expression analyses identified four potential miR-222 targets (p27, Hipk1, Hipk2, and Hmbox-1) in CMs. These were confirmed as direct targets by luciferase assays, site-directed mutagenesis, and immunoblotting. siRNA knockdown (KD) of p27 or Hipk1 induced neonatal CM proliferation, while siRNA KD of Hmbox-1 increased CM size.

To examine miR-222’s role in vivo, LNA-antimiR-222 was injected via tail vein or subcutaneously and shown to reduce cardiac miR-222 levels to 1.5% (p<0.01). Untreated animals subjected to three weeks of swimming had the expected increase in heart size (15% in HW/TL, p<0.05), CM size (26%, p<0.05), and markers of CM proliferation (Ki67 and pHH3, p<0.05). Increases in heart and CM size were unaffected by control LNA-antimiR but completely blocked by LNA-antimiR-222, while CM proliferation markers decreased (60%, p<0.05). To see if miR-222 is sufficient to mediate the benefits of exercise, we made transgenic mice with cardiac-specific, regulated expression of miR-222 (Tg-miR-222). Tg-miR-222 have normal heart size and function at baseline. After ischemia-reperfusion injury (IRI), Tg-miR-222 had similar initial dysfunction but were protected against adverse remodeling over the next six weeks with better function (p<0.01), less cardiac fibrosis (68%, p<0.05), and increased CM proliferation markers (63%, p<0.05).

Conclusion:
Cardiac miR-222 is upregulated by exercise, necessary for exercise-induced cardiac growth, and protects against pathological cardiac remodeling after IRI.
Abstract 254: Small Molecule Induced Caveolin Regulate GPCR Signaling In IPS cells Survival And Differentiation
Authors: PASHA ZEESHAN , ASHRAF MUHAMMAD
Published: July 2021

Background:
Previously we reported the use of a small molecule in upregulating cardiac genes by GPCR signaling. We further investigated the small molecule mediated effect involve caveolin and critical miRs that modulate the signaling pathway for increased survival reduced apoptosis and cardiac regeneration in ischemic myocardium.

Methods and Results:
IPs were treated with a small molecule (ZP-G;20uM) for three to five days. Increased cell survival and proliferative activity (p<0.01 vs non treated IPS) with reduced cytochrome c release in cytosol was observed in small molecule treated IPS. Given the upregulation of cardiac genes, ultrastructure feature of small molecule treated IPS cells showed caveolin formation on transmission electron microscopy. miR Microarray analysis of treated IPS cells showed upregulation of miR 705,149, cardiac specific miR −133,762 and down regulation of miR-16, miR 34a, miR-290/292-5p modulating the downstream targets Chemokines, VEGFA, Vesicles associated membrane protein (Vamp), Cdk, E2f, Wnt3a and GPCR receptors. mRNA Microarray further validate 2 fold downregulation of Dnmt1,3b, Max gene associated protein, CDK2, NFkB repressing factor with concomitant 2-3 fold upregulation of caveolin, chemokines and integrins. Western blot analysis displayed significant upregulation of Gα protein, phospho Akt, and Bcl-2 levels as compared to nontreated IPS cells. Small molecule mediated effect was abolished by treatment of IPS cells with GPCR blocker. No tumor formation when treated IPS cells were injected in nude mouse. For Invivo study, female mice with myocardial infarction after permanent LAD ligation were transplanted with either DMEM (Gp1),or 2х105 non treated IPS (Gp 2), or treated IPS (Gp 3). At 4-week of engraftment of IPS cell derived cardiac progenitors (Gp 3) showed reduced apoptosis, extensive survival and myogenesis with attenuation of infarct size and improved heart function as compared to other groups.

Conclusion:
This study provides the first evidence that small molecule differentiate IPS to cardiac progenitors and regenerate the ischemic myocardium by targeting downstream targets that regulate the transcription of critical genes and proteins expression to facilitate receptor- tissue- cell signaling.
Abstract 258: Pitx2 Promotes Murine Myocardial Regeneration after Myocardial Injury
Authors: TAO GE , Klysik Elzbieta , Morikawa Yuka , Martin James F
Published: July 2021

Myocardial infarction is the leading cause of morbidity and mortality in the United States. Compromised myocardial function, due to the lack of self-renewal capacity in mature hearts, is a major reason for heart failure. Available therapies can only ameliorate, but not reverse the loss of functional myocardium. With heart transplantation as the only available cure, design of an effective regenerative therapy has become imperative for cardiovascular research. To repopulate the heart with de novo cardiomyocytes, most attempts have been based on the transplantation of cardiac, non-cardiac stem cells or their derivatives, however a more profound knowledge of stem cells is required for achieving significant progress. Meanwhile, triggering endogenous regenerative capacity is a compelling strategy for cardiac repair. It has been reported that proliferation of pre-existing cardiomyocytes strongly contributes to regeneration. Thus, efforts have been made to reintroduce mature cardiomyocytes into mitotic cycle. The mechanisms underlying the proliferation of cardiomyocytes during development and their homeostasis during adulthood are not fully understood, but likely require tight regulation of transcription factors in specific cell types. We have previously shown that the mouse Hippo kinase cascade is a major heart-size control pathway during development. In addition, activation of Yap, a transcriptional cofactor inhibited by Hippo, by genetically disrupting Hippo signaling is sufficient to induce juvenile and adult myocardial regeneration after surgery-induced myocardial infarction. Here we identified the paired-like homeodomain transcription factor 2 (pitx2) as a potential downstream target and cofactor of Yap in mouse heart. Our data indicates that Pitx2 expression is induced by myocardial injury, and is required for neonatal myocardial regeneration in a postnatal day 1 (P1) apex resection model. Further studies show that over-expression of pitx2 in adult cardiomyocytes is sufficient to promote the restoration of myocardial structure and function after myocardial infarction. Together, we show that pitx2 is a new manipulator of myocardial regeneration and could serve as a novel therapeutic target in cardiac regenerative therapy.
Abstract 273: Glycogen Depletion During Hypoxia Results in Enhanced AMPK Phosphorylation and Diminished Smooth Muscle Contractility in Human Saphenous Vein
Authors: Segar Lakshman , Pyla Rajkumar , Pichavaram Prahalathan , Fairaq Arwa , Kozak Mark , Kamath Vinayak , Patel Vijay
Published: July 2021

Objectives:
Hypoxia promotes vasodilation of coronary arteries as a protective response to improve blood flow to the myocardium. Although diminished formation of ATP and enhanced phosphorylation/activation of AMP-activated protein kinase (AMPK) in vascular smooth muscle are known to mediate hypoxia-induced vasorelaxation, the contribution of glycogen toward AMPK phosphorylation and contractility remains unclear.

Methods:
Using surgically-removed endothelium-denuded human saphenous vein segments ex vivo, the present study has examined serotonin (5-HT)-induced smooth muscle contractility by isometric tension measurements and AMPK phosphorylation by immunoblot analysis under normoxic (95% O2/5% CO2) and hypoxic conditions (95% N2/5% CO2, 30 min).

Results:
Under normoxic glycogen-enriched conditions, the maximal contractile response (Emax) and sensitivity (pEC50) to 5-HT-induced contractility were 145 ± 3% and 6.7 ± 0.2, respectively (n = 4). Induction of hypoxia diminished the glycogen content by ~95% (3.7 ± 1 under hypoxia versus 73.6 ± 5 μg/mg protein under normoxia; n = 2). Importantly, glycogen depletion led to diminution in 5-HT-induced maximal contractility to 37 ± 11% with an accompanying exaggerated increase in AMPK phosphorylation, compared with normoxic conditions (n = 4). Inclusion of exogenous D-glucose (5.5 mM) prevented the exaggerated increase in AMPK phosphorylation thereby restoring 5-HT-induced maximal contractility to 125 ± 6% (n = 4). Parallel studies that included either L-glucose or 2-deoxy-D-glucose (non-metabolizable forms of glucose) did not show any changes in contractility or AMPK phosphorylation.

Conclusion:
The present findings suggest that depletion of smooth muscle glycogen during hypoxia may limit the availability of intracellular glucose in vein grafts, thereby enhancing AMPK phosphorylation to promote vasorelaxation.
Abstract 277: Insights Into The Transcriptional Reprogramming Induced By Beta-3 Adrenergic Cardioprotection
Authors: Garcia-Prieto Jaime , Sanz-Rosa David , Sanchez-Cabo Fatima , Dopazo Ana , Fuster Valentin , Ibanez Borja
Published: July 2021

Background:
Beta-3 adrenergic receptor (β3AR) stimulation reduces myocardial ischemia/reperfusion injury (IRI) in vivo. The molecular mechanisms that regulate this cardioprotective therapy are a matter of intense research. The aim of this study was to assess the transcriptional reprogramming underpinning cardioprotection.

Methods and Results:
HL-1 cardiomyocytes were subjected to 6 hours of hypoxia followed by 18h of reoxygenation (H/R) in the presence or absence of the beta-3 adrenergic receptor agonist BRL37344 (5μM). As expected, H/R caused cardiomyocyte death, which was significantly supressed by beta-3 agonist treatment. In order to uncover the molecular basis of cytoprotection we analysed gene expression profiles in HL-1 cells subjected to hypoxia (6h) and reoxygenation (1h, 6h). Immediate reoxygentation proufoundly altered the hypoxia gene expression signature at 1h but this effect was lost upon extended reoxygenation. BRL treatment was able to reverse transcriptomic alterations associated with increased cell death, by supressing the dysregulation of cell survival pathways (possibly implicating mTORC, EGF, eNOS, JAK/Stat, and IL-10 signalling.)

Conclusion:
Beta-3-adrenergic stimulation induces an adaptive transcriptional reprogramming that preserves cell viability by converging a variety of cytoprotective pathways.
Abstract 279: A Deletion In The N2A Region Of Titin Carried By Muscular Dystrophy With Myositis (mdm) Mice Severely Affects Skeletal Muscle, But Not The Heart
Authors: Lunde Ida G , Wakimoto Hiroko , Burke Michael A , Linke Wolfgang , Christensen Geir , Seidman Jonathan G , Seidman Christine E
Published: July 2021

20% of dilated cardiomyopathy patients carry mutations in the giant protein titin. Mutations are predominant in A band but also occur in I band, a domain that regulates passive tension and myocyte signaling. A recessive mouse mutation in titin I band N2A region (mdm) causes early onset muscular dystrophy with myositis and death. We assessed cardiac morphology, function, and transcriptional profiles (RNAseq) in mdm mice.

Young homozygous mdm mice (n>6) have reduced body weight (7gms) vs. heterozygous (20gm) or WT (17gm) littermates, with severe skeletal muscle dystrophy. Four-week old homozygous mdm mice have higher left ventricular (LV): body weight ratios. Echocardiography revealed thinner LV posterior wall and septum (LVPWd and IVSd) and normal LV diameter (LVDd); when normalized for body weight, cardiac dimensions were increased compared to WT or heterozygous mdm mice. Fractional shortening was reduced in homozygous Mdm mice (35%) vs. WT (40-41%, p<0.01); histology showed neither overt pathology nor fibrosis. Titin gels showed lack of difference in cardiac titin isoform pattern, consistent with RNAseq, which showed the mdm titin transcript excluded exons 107 and 108, deleting in frame 48 amino acids. 240 transcripts (0.8%) were differentially expressed (fold change >1.5 and <0.75, p<0.001) in homozygous vs. heterozygous mdm hearts; ANP and BNP were mildly upregulated (2- and 1.2-fold). Altered transcripts participated in extracellular and immune signaling pathways. Among titin binding partners, only calpain-3 that interacts with N2A was changed (0.6-fold), consistent with previous reports in skeletal muscle. As humans have heterozygous mutations, we stressed adult heterozygous mdm and WT mice (2 weeks of angiotensin II infusion): both had comparable hypertrophic responses (increased LVPWd and IVSd). Aged (89 week old) unstressed heterozygous mdm mice had normal cardiac dimensions and function.

The N2A region, I-band titin mdm mutation causes minimal cardiac dysfunction in mice, unlike the severe skeletal muscle phenotype. Human I-band mutations are unlikely to cause dilated cardiomyopathy.
Abstract 294: Decreased Plasma Omega-3 to Omega-6 Polyunsaturated Fatty Acid Ratio Associated with Apical Ballooning Syndrome (Tako-Tsubo cardiomyopathy -TTC)
Authors: Katoh Youichi , Nagamine Show , Wada Takeshi , Isogai Hiroyuki , Ozaki Dai , Shimai Ryosuke , Kakihara Midori , Yuu Hiroshige , Yokoyama Ken , Oigawa Tetsuya , Yaginuma Kenji , Nakazato Yuji
Published: July 2021

Background:
Eicosapentaenoic acid (EPA) of the omega-3 polyunsaturated fatty acids (ω-3 PUFA) family plays important roles in the prevention of cardiovascular disease, while, arachidonic acid (AA) of the ω-6 PUFA family promotes inflammatory and prothrombotic influences. Tako-Tsubo cardiomyopathy (TTC) is a heart syndrome associated with transient myocardial contractile dysfunction. Decreased endothelial function in response to acute mental stress has been reported in patients with a prior episode of TTC. However, the pathogenesis of TTC remains unclear and the relationship between TTC and EPA/AA ratio has not been elucidated.

Methods and Results:
This study consisted of 10 consecutive patients with clinically diagnosed TTC. The aim of this study was to investigate the association between the plasma EPA/AA ratio and the early stage of clinically diagnosed TTC. To examine the plasma fatty acid level, blood samples were obtained from control, old myocardial infarction, and clinically diagnosed TTC patients. A clinically diagnosed TTC patients revealed a lower plasma EPA/AA ratio [Control patients, 0.58 (n=18) vs OMI patients, 0.38 (n=22) vs TTC patients, 0.12 (n=10), p=0.011]. High-sensitivity CRP levels and a low plasma EPA/AA ratio could independently predict the prevalence of TTC on multivariate logistic regression analysis [odds ratio 1.83 (95%CI 1.03-3.25), p=0.036 and odds ratio 2.05 (95%CI 1.12-3.92), p=0.02)].

Conclusion:
In patients with TTC, a low plasma EPA/AA ratio was significantly associated with the early stage of clinically diagnosed TTC. The findings implicate lower EPA/AA ratio and the following endothelial dysfunction as a potential mechanism involved in the pathogenesis of this unique cardiomyopathy.
Abstract 295: Cardiac-Specific Gene Leucine-Rich Repeat Containing 10 (Lrrc10) is required for proper Cardiac Contractility and Responses to Biomechanical Stress
Authors: Lee Youngsook , Brody Matthew , Feng Li , Reynolds Courtney , Balijepalli Ravi
Published: July 2021

The molecular genetic basis for early onset dilated cardiomyopathy (DCM) is largely unknown. We have identified that Lrrc10 is a cardiac-specific gene regulated by Nkx2.5 and GATA4. Lrrc10-null (Lrrc10-/-) mice exhibit prenatal systolic dysfunction followed by early onset DCM after birth. However, mechanistic roles for LRRC10 in the regulation of cardiac pathophysiology are unknown. We hypothesize that LRRC10 expression is critical for myocyte contractility and proper responses to pressure overload. Various mouse models with pathological cardiac hypertrophy showed decreased levels of Lrrc10. To test roles of LRRC10 in response to biomechanical stress, transverse aortic constriction (TAC) was performed. Lrrc10-/- mice showed significant increases in left ventricular inner diameter dimensions and drastically reduced % fractional shortening in response to 4 weeks of TAC (13%) after 4 weeks, while no significant changes occurred in WT after TAC (26% F.S.). These data indicate that pressure overload greatly exacerbates systolic dysfunction in Lrrc10-/- vs WT hearts. Next, the role of LRRC10 in cardiac excitation contraction coupling was investigated. Myocyte contractility and calcium transients were studied using the video-edge detection system and fura 2-AM loading at basal levels and following perfusion with Isoproterenol (ISO, 10 nM) at 0.5 - 2 Hz pacing. Cell shortening was significantly reduced following perfusion with ISO in Lrrc10-/- myocytes (55% and 49% at 1 and 2 Hz, respectively) vs WT. Calcium transient analysis showed that there were no differences in peak amplitudes of [Ca2+]i between Lrrc10-/- and WT myocytes at baseline or with ISO treatment. Whole-cell voltage clamp experiments demonstrated a significant reduction in the L-type Ca2+ current (ICa,L) density in Lrrc10-/- myocytes (-2.5 ± 0.2 pA/pF) compared to WT myocytes (-6 ± 0.6 pA/pF). The inactivation of the ICa,L in Lrrc10-/- myocytes was significantly delayed. Further, co-immunoprecipitation showed that LRRC10 directly associated with Cav1.2 subunit of LTCC, which correlates well with the localization of LRRC10 to dyadic regions in adult cardiomyocytes by TEM. Therefore, LRRC10 is a novel DCM candidate gene essential for normal myocyte contractility via regulating ICa,L.
Abstract 3: Il-10 Regulated Mir-375 Enhances Endothelial Progenitor Cell Mediated Myocardial Repair And Survival After Myocardial Infarction.
Authors: Garikipati Venkata N , Krishnamurthy Prasanna , Verma Suresh K , Mackie Alexandra R , Vaughan Erin E , Khan Mohsin , Ramirez Veronica , Abramowa Tatiana , Ellingson Kenneth L , Misener Sol , klingler Aiko I , Qin Gangjian , Koch Walter J , Kishore Raj
Published: July 2021

We hypothesized that IL-10 regulates miR-375 signaling in EPCs to enhance their survival and function in ischemic myocardium after MI. miR-375 knock down EPC were transplanted intramyocardially after induction of MI. Mice receiving EPC treated with miR-375 inhibitor showed increased number of GFP+EPCs retention that was associated with reduced EPC apoptosis in the myocardium. The engraftment of EPC into the vascular structures and the associated capillary density was significantly higher in miR-375-treated mice. The above findings further correlated with reduced infarct size, fibrosis and enhanced LV function (echocardiography) in miR-375 knock down EPC group as compared to scrambled EPC. Our in vitro studies revealed that the knockdown of miR-375 enhanced EPC proliferation, migration; tube formation ability and inhibited cell apoptosis, while the up-regulation of miR-375 with the mimic had the opposite effects. In addition, we found that miR-375 negatively regulates the expression of 3-phosphoinositide-dependent protein kinase 1 (PDK1) by directly targeting the 3'UTR of the PDK1 transcript. Interestingly, EPC isolated from IL-10-deficient mice has elevated basal levels of miR-375 and exhibited poor proliferation and tube formation ability where as miR-375 knock down in EPC isolated from IL-10 deficient mice attenuated these effects. Furthermore, transplantation of miR-375 knock down IL-10 deficient EPC after MI resulted in attenuated cardiac functions compared to scramble IL-10 deficient EPCs. Taken together, our studies suggest that IL-10 regulated miR-375 enhances EPC survival and function, associated with efficient myocardial repair via activation of PDK-1/AKT signaling cascades.
Abstract 309: Small Heterodimer Partner Blocks Cardiac Hypertrophy By Interfering With GATA6 Signaling
Authors: Nam Yoon Seok , Kwon Duk-Hwa , Eom Gwang Hyeon , Kook Hyun
Published: July 2021

Rationale:
Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA binding domain. By interacting with other transcription factors, SHP regulates diverse biological events including glucose metabolism in liver. The role of SHP in adult heart diseases has not yet been demonstrated.

Objective:
We aimed to investigate the role of SHP in adult heart in association with cardiac hypertrophy.

Methods and Results:
The roles of SHP in cardiac hypertrophy were tested in primary cultured cardiomyocytes and in animal models. SHP null mice showed a hypertrophic phenotype. Hypertrophic stresses repressed the expression of SHP, whereas forced expression of SHP blocked the development of cardiomyocyte hypertrophy. SHP reduced the protein amount of Gata6. By direct physical interaction with Gata6, SHP interfered with the binding of Gata6 to GATA binding elements in the promoter regions of natriuretic peptide precursor type A. Metformin, an anti-diabetic agent, induced SHP and suppressed cardiac hypertrophy. The metformin-induced anti-hypertrophic effect was attenuated either by SHP siRNA in cardiomyocytes or in SHP null mice.

Conclusions:
These results establish SHP as a novel anti-hypertrophic regulator that acts by interfering with GATA6 signaling. SHP may participate in the metformin-induced anti-hypertrophic response.
Abstract 319: Small Molecule Gβγ Inhibition Attenuates Cardiac Fibroblast Inflammatory and Pro-Fibrotic Signaling
Authors: Travers Joshua G , Kamal Fadia A , Burhans Michael S , Blaxall Burns C
Published: July 2021

Heart failure (HF) is a devastating disease characterized by chamber remodeling, interstitial fibrosis and reduced ventricular compliance. Prolonged sympathetic overstimulation promotes excess signaling through G-protein Gβγ subunits and ultimately results in pathologic GRK2-mediated β-adrenergic receptor (β-AR) downregulation. We have recently demonstrated the therapeutic potential of the small molecule Gβγ-GRK2 inhibitor Gallein in limiting HF progression. Pathologic activation of the cardiac fibroblast (CF) induces the transition to a myofibroblast phenotype, which plays a fundamental role in myocardial fibrosis and remodeling. We hypothesized that Gβγ-GRK2 inhibition plays an important functional role in the CF to attenuate pathologic CF activation, inflammation and interstitial fibrosis.

To explore the effect of Gβγ-GRK2 inhibition on inflammation and pro-fibrotic signaling, mice were subjected to 7 days of transverse aortic constriction, a pressure-overload model of HF. In addition to the attenuation in overall cardiac hypertrophy, animals treated with Gallein displayed reduced expression of pro-inflammatory cytokines, including macrophage inflammatory protein 1 alpha (MIP-1α) and MIP-1β, along with Interleukin-6, as assessed by qPCR. Gallein-treated animals also exhibited diminished pro-fibrotic signaling, as evidenced by a reduction in the expression of TGFβ, a major driver of myocardial fibrosis, and decreased expression of collagen. To recapitulate these findings in vitro, primary adult mouse ventricular fibroblasts were pathologically stimulated using Isoproterenol (ISO, β-AR agonist) or Angiotensin II and treated +/- Gallein for 24 hours. CFs treated with Gallein displayed an analogous reduction in the expression of these pro-inflammatory cytokines and collagen.

In summary, these data suggest a potential therapeutic role for small molecule Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, inflammation and interstitial fibrosis. We believe this fibroblast-targeted approach will lead to the refinement of existing targets and compounds, and possibly the generation of novel therapeutics for the treatment of HF.
Abstract 322: Metformin Inhibits Mitochondrial Permeability Transition Pore Opening in Human Cardiac Mitochondria
Authors: Emelyanova Larisa , Gudlawar Sirisha , Cosic Milanka , Mirza Mahek , Rizvi Frahan , Holmuhamedov Ekhson , Downey Francis X. , Tajik A. Jamil , Jahangir Arshad
Published: July 2021

Background:
Type II diabetes mellitus is a major health problem contributing to increased morbidity and mortality with associated cardiovascular diseases. Metformin, an oral antidiabetic agent, has cardioprotective properties independent of their glucose lowering effect; however, mechanisms underlying cardioprotection remain poorly defined. We hypothesized that the cardioprotective effect of metformin appears to be associated with inhibition of mitochondrial permability transition pore (mPTP) that could be beneficial in diabetic hearts with attenuated endogenous cardioprotective responses.

Purpose:
The aim of the study was to determine the protective effect of metformin on mPTP opening in mitochondria from human myocardium..

Methods:
Mitochondria freshly isolated from the left atrial appendage of nondiabetic patients undergoing cardiac surgery were loaded with calcium- (fluo-3) and membrane potential-sensitive (Safranin-O) fluorescent dyes and challenged with sequential pulses of Ca2+ (10 μM every 3 m) in the absence and presence of different concentrations of metformin (2.5, 5, 10 mM). The sensitivity of mitochondria toward mPTP opening was assessed by abrupt release of mitochondrial Ca2+ (fluo-5N fluorescence), with simultaneous dissipation of mitochondrial membrane potential (safranin O fluorescence) and mitochondrial swelling (decrease in light scattering).

Results:
Metformin caused a dose-dependent inhibition of Ca2+-induced mPTP opening with delayed mitochondrial Ca2+ release, depolarization and swelling. The tolerance of metformin-treated mitochondria to Ca2+-induced mPTP opening when compared to controls was increased from 457±71 to 600±74 nmol/mg protein at 2.5mM, 674±10 nmol/mg protein at 5 mM and 750±77 nmol/mg protein at 10 mM. Cyclosporin A (1 μM), a known mPTP inhibitor, has marginal incremental effect on metformin-induced inhibition of mPTP opening.

Conclusion:
In human cardiac mitochondria, metformin inhibited calcium-overload-mediated mPTP opening that otherwise leads to mitochondrial energetic failure and cell death. Thus, metformin may help restore attenuated cardioprotection in diabetic hearts by increasing tolerance to stress-induced mPTP opening and, thus, preventing cell death.
Abstract 324: Dronedarone Alters the Sensitivity of Cardiac Mitochondria to Permeability Transition Pore Opening
Authors: Gudlawar Sirisha , Cosic Milanka , Rizvi Farhan , Holmuhamedov Ekhson , Thakur Monika , Mirza Mahek , Tajik Jamil , Dawney Francis , Jahangir Arshad
Published: July 2021

Introduction:
Dronedarone (DR) is a new antiarrhythmic agent that was design to minimize side effects of Amiodarone. However, in clinical trials was shown to worsen heart failure (HF) and increased mortality. The mechanisms for these adverse effects are not known. Since mitochondria are critical for maintaining cardiac energetics and regulate, we hypothesized that DR increases sensitivity towards opening of mitochondrial permeability transition pore (mPTP) that underlies mitochondrial failure and cell death.

Methods:
Mitochondria were isolated from left atrial appendage tissue of patients undergoing bypass cardiac surgery (n=3). Patients did not have any history of HF or atrial fibrillation. To induce mPTP, mitochondria were exposed with sequential additions of Ca
2+
(Fluo-5N) in the presence of 5, 10, 20 μM of DR. The opening of mPTP was determined by monitoring abrupt release of Ca
2+
, rapid loss of membrane potential (ΔΨ) and mitochondrial swelling.

Results:
DR accelerated Ca
2+
-induced mPTP opening starting at 5 μM concentration (Fig). At concentration of 20 μM, it completely depolarized mitochondria abolishing capacity for Ca
2+
handling and responsiveness to cyclosporine A (CsA), an inhibitor of mPTP.

Conclusion:
In isolated human cardiac mitochondria from patients without history of HF DR at clinically relevant concentration increased sensitivity of mitochondria to Ca
2+
induced mPTP opening. In failing heart this can be compromised and needs to be studied.

Dronedarone accelerates Ca
2+
-induced mPTP opening in mitochondria isolated from human left atrial appendage.
Abstract 329: Simvastatin Inhibits TGF-β1-induced Proliferation and Activation of Cardiac Fibroblasts Through Inhibition of SMAD Pathway
Authors: Rizvi Farhan , Siddiqui Ramail , DeFranco Alesandra , Jayaprakash Akhil , Mirza Mahek , Emelyanova Larisa , Ross Gracious , Hulmohamedov Ekhson , O’Hair Daniel , Tajik A. Jamil , Jahangir Arshad
Published: July 2021

Background:
HMG-CoA reductase inhibitors (statins) have been shown to reduce the incidence of atrial fibrillation (AF) and its progression but the underlying mechanisms are not fully elucidated. Since atrial fibrosis plays a major role in the development of the substrate for AF progression, we hypothesized that statins antagonize the effect of profibrotic cytokines, reducing their stimulatory effect on fibroblast proliferation, differentiation and activation.

Methods:
The effect of TGF-β1, a major profibrotic cytokine, on fibroblast proliferation and activation of myofibroblasts was assessed by expression of alpha smooth muscle actin (α-SMA) message (qPCR) and proteins (western and immunofluorocytochemistry) in the presence and absence of simvastatin (1-10μM). The inhibitory effect of simvastatin on SMAD 2/3 phosphorylation (western) and its nuclear translocation by TGF-β1 (5ng) was determined by immunofluorescence antibodies using fluorescent microscopy.

Results:
TGF-β1 treatment increased fibroblast proliferation (cell count) by 63% compared to control (p<0.001) at 96 hours, which was inhibited by simvastatin by 61% (p<0.001) (Fig a). Simvastatin also reduced TGF-β1-mediated myofibroblast differentiation (α-SMA positive) by 75% (p=0.02); (Fig b and c). TGF-β1 increased SMAD2/3 phosphorylation with increase in nuclear localization was inhibited by simvastatin.

Conclusion:
Simvastatin inhibits TGFβ-1-mediated cardiac fibroblast proliferation and myofibroblast differentiation by antagonizing SMAD phosphorylation and its translocation into the nucleus.
Abstract 33: Contractile Dysfunction In The Mouse Heart Caused By Phospholipase C beta1b Mediated Activation Of Protein Kinase Calpha
Authors: Grubb David R , Ma Yi , Luo Jieting , Crook Bryony , Cooley Nicola , Kiriazis Helen , Qian Hong Wei , Gregorevic Paul , Gao Xiao-Ming , Du Xiao-Jun , Woodcock Elizabeth A
Published: July 2021

The activity of the early signaling enzyme, phospholipase Cβ1b (PLCβ1b), is elevated in diseased myocardium and activity increases with disease progression. PLCβ1b and the alternative splice variant, PLCβ1a, were expressed in mouse hearts using adeno-associated viral constructs (rAAV6-FLAG-PLCβ1b, rAAV6-FLAG- PLCβ1a) delivered intravenously. Functional responses were assessed in vivo and confirmatory mechanistic studies were conducted in neonatal rat ventricular myocytes (NRVM). FLAG-PLCβ1b was expressed in all of the chambers of the mouse heart, but was highest in left ventricle, where expression was observed in >90% of the cells and was localized to the sarcolemma and T-tubules. Heightened PLCβ1b expression caused a rapid loss of contractility and down-regulation of Phospholamban expression. The loss of contractility induced by PLCβ1b was reversed by inhibition of protein kinase Cα (PKCα). PLCβ1a did not affect contractile function or phospholamban expression. Mechanistic analysis performed in neonatal rat cardiomyocytes confirmed PLCβ1b increased the membrane association of PKCα as well as downstream dephosphorylation of phospholamban and depletion of the Ca2+ stores of the sarcoplasmic reticulum, both of which were mediated by PKCα. Trans-aortic constriction (TAC) resulted in progressive hypertrophy together with reduced contractility in PLCβ1a expressing mice. In PLCβ1b-expressing mice, TAC induced a similar hypertrophic response, but did not cause further contractile depression above that due to PLCβ1b expression alone, suggesting that PLCβ1b is responsible for lowering contractility in response to pressure overload. We conclude that heightened PLCβ1b activity observed in diseased myocardium contributes to pathology by PKCα-mediated contractile dysfunction. PLCβ1b is a cardiac-specific signaling system, and thus provides an ideal therapeutic target for the development of well-tolerated inotropic agents for use in failing myocardium.
Abstract 330: Metallothionein Preservation Of Cardiac Akt2 Signaling By Down-regulating Trb3 Prevents Diabetic Cardiomyopathy
Authors: TAN YI , Yan Xiaoqing , Zhou Shanshan , Li Yong , Li Yan , Cai Lu
Published: July 2021

Cardiac insulin resistance is a key pathogenic factor for diabetic cardiomyopathy, but its mechanism remains largely unclear. Here we demonstrated that diabetes significantly inhibited cardiac Akt phosphorylation from 2 weeks to 2 months in wide-type (WT) mice, but not in cardiac-specific metallothionein-transgenic (MT-TG) mice. Cardiac Akt2 expression and phosphorylation was decreased and insulin-induced cardiac Akt2 and GSK-3β phosphorylation and glycogen synthase dephosphorylation were also decreased in WT, but not MT-TG, diabetic mice. Deletion of the Akt2 gene either in vitro H9c2 cells or in vivo significantly impaired cardiac glucose metabolic signaling. In addition, diabetes significantly increased cardiac Akt negative regulator tribbles (TRB)3 expression only in WT mice, suggesting the possible contribution of MT inhibition of diabetic up-regulation of TRB3 to Akt2 function preservation. Cardiac H9c2 cells with and without forced MT-overexpression (MT-H9c2) were treated with tert-butyl hydroperoxide (tBHP), which significantly reduced Akt2 phosphorylation in both basal and insulin-stimulating conditions only in H9c2 cells. Silencing TRB3 expression with SiRNA completely prevented tBHP’s inhibition of insulin-stimulated Akt2 phosphorylation in H9c2 cells, while overexpression of TRB3 in MT-H9c2 cells completely abolished MT preservation of insulin-stimulated Akt2 phosphorylation. Forced-overexpression of TRB3 by adenovirus-mediated gene delivery in MT-TG hearts also abolished MT’s preservation of cardiac insulin signaling and prevention of diabetic cardiomyopathy. These results suggest that diabetes-attenuated cardiac Akt2 function via up-regulating TRB3 plays a critical role in diabetic inhibition of insulin signaling in the heart. MT preserved cardiac Akt2-mediated insulin signaling by inhibiting TRB3, leading to the prevention of diabetic cardiomyopathy.
Abstract 331: Recovery of Mitochondrial Bioenergetics after Hypoxia via Regulating Pyruvate Dehydrogenase Kinase and Adenosine Monophosphate-activated Kinase
Authors: Toli Jessica M , He Minzhen , Suzuki Carolyn , Abdellatif Maha
Published: July 2021

Mitochondrial quality control is critical for the survival of cardiac myocytes during stress. The purpose of this study was to examine the effect of metabolic substrates and regulators of metabolism on mitochondrial bioenergetics, as an indicator of mitochondrial quality, and how these factors might influence the recovery of the cell’s bioenergetics after hypoxia/ischemia. By monitoring oxygen consumption rates (OCR), in real-time, in live neonatal rat myocytes and human cardiac myocyte-differentiated induced pluripotent stem cells, we found that both cell types can maintain basal OCR efficiently with any metabolic substrate; however, the neonatal cells require both glucose and fatty acid, while the human adult cells require fatty acid only, for mounting maximum reserve respiratory capacity (RRC). Our data also show that subjecting cardiac myocytes to hypoxia results in a reduction of the cells’ basal OCR and oxidative phosphorylation, and exhausts the RRC, which is accompanied by an increase in pyruvate dehydrogenase kinase (Pdk) 1 and 4. Except for normalization of Pdk1 levels, there was little or no recovery of these parameters after reoxygenation. We, thus, hypothesized, that inhibition of Pdks may help recovery of the cell’s bioenergetics. Indeed, our results show that by inhibiting Pdks with dichloroacetate (DCA) before or after hypoxia, the cells’ bioenergetics, including OCR, oxidative phosphorylation, and RRC in neonatal myocytes, and RRC in the human myocytes fully recover within 24 h. On the other hand, activating AMP-activated kinase (AMPK) resulted in delayed (96 h) improvement of the cells’ RRC that was accompanied by an increase in peroxisome proliferator-activated receptor gamma, coactivator 1α (3.5x), peroxisome proliferator-activated receptor-α (2x), and mitochondrial number (2x). These results led us to conclude that compromised mitochondrial quality can be rescued through mechanisms that regulate glucose or fatty acid oxidation by either inhibiting Pdks or activating AMPK, respectively, in rodent and human myocytes.
Abstract 348: Multi-Species Genome-Wide Analyses of the Specification of Individual Cardiac Cell Fates
Authors: Busser Brian , Haimovich Julian , Chen Guokai , Ovcharenko Ivan , Michelson Alan
Published: July 2021

There are remarkable molecular and embryological similarities in cardiogenesis between Drosophila and vertebrates. Cells comprising the Drosophila heart can be subdivided into individual identities based on differences in morphology, function and gene expression patterns. Recent studies have shown that differential modifications of histone proteins, in vivo transcription factor (TF) binding, and the presence of particular TF binding motifs can be used as predictive signatures of the enhancers that govern cell-specific gene expression. Here we used discriminative training methods within an integrative, multi-species framework to uncover the motifs, enhancers and genes underlying cardiac cell fate decisions. As an initial step, we undertook a large-scale validation of Drosophila heart enhancers, which revealed enhancer activities in distinct subpopulations of cardiac cells. To identify related cell-specific regulatory elements, we used the validated enhancers as a training set in a machine learning approach that integrated TF motifs with ChIP data for both TF binding and histone modifications. Empirical validation of candidate enhancers predicted by this method confirmed activity in the appropriate cardiac cells. By clustering the motifs derived from the individual cardiac classifiers, we identified and validated sequence features which discriminate specific cellular identities. Next, we asked if similar predictive signatures underlie mouse and human cardiomyocyte (CM) differentiation from embryonic stem cells (ESCs). We show that the distribution of histone marks found within differentiating human and mouse ESCs indeed predict genes potentially critical for CM differentiation, with the best predictions provided by the overlapping mouse and human candidates. We evaluated this result in a large-scale RNAi-based screen of Drosophila orthologs of the mammalian genes, which uncovered dozens of novel cardiogenic regulators whose function is being tested in differentiating human ESCs. In total, these results document the utility of computational modeling combined with empirical testing to uncover the enhancers, TF motifs and genes which characterize individual cardiac cell fates in both invertebrate and mammalian species.
Abstract 35: 2-deoxy Adenosine Triphosphate Improves Contraction In Human End-stage Heart Failure
Authors: Moussavi-Harami Farid , Razumova Maria V , Cheng Yuanhua , Racca Alice W , Stempien-Otero April , Regnier Michael
Published: July 2021

We are developing a novel approach to treat heart failure that involves adeno-associated virus vectors to elevate intracellular 2 deoxy-ATP (dATP) via increased expression of the enzyme Ribonucleotide Reductase. Our studies in rodents have shown that substitution of dATP for ATP as the energy substrate increases contraction in striated muscle. Here we report for the first time the effect of dATP on human cardiac muscle contraction. We measured the contractile properties of demembranated multicellular ventricular wall preparations and isolated myofibrils from adult human heart tissue obtained from twelve patients undergoing surgery for cardiac transplantation or placement of left ventricular-assist device for end-stage heart failure. Isometric force at saturating calcium concentration was increased by about ten percent from 38.6±3.8 mN/mm2 to 42.8±4.2 mN/mm2 when dATP was substituted for ATP (P<0.001). The effect was even greater at physiologic calcium concentrations with an approximate increase of thirty percent. Isometric force increased from 21.2 ± 6.1 mN/mm2 to 26.4± 6.4 mN/mm2 (p<0.001) at pCa=5.6 and from 22.6 ± 5.9 mN/mm2 to 27.5± 6.3 mN/mm2 (p<0.001) at pCa=5.8 with dATP for ATP substitution. The result was an increase in the Ca2+ sensitivity of force as the [Ca2+] required to elicit half maximum force (pCa50) increased by 0.08 units from 5.68±0.03 to 5.79±0.03 (N=24, P< 0.001). The maximum rate of force redevelopment (ktr) in demembranated wall muscle increased (0.82±0.01 s-1 vs. 0.62±0.01 s-1, P< 0.05), as was the rate of contractile activation in isolated myofibrils (0.80±0.06 s-1 vs. 0.57±0.06 s-1, P<0.01) suggesting dATP may increase dP/dT in failing human myocardium. Importantly, there was no slowing of relaxation, as the time to 50% and 90% myofibril relaxation were unchanged. Purified myosin from failing human myocardium showed enhanced NTPase activity with dATP (0.89±0.17 s-1/head) vs. ATP (0.55±0.20 s-1/head, P<0.05). In conclusion, the data strongly suggest dATP increases cross-bridge cycling, compared with ATP in failing human myocardium and shows promise in restoring cardiac pump function. These data support a novel myofilament approach for treating heart failure that warrants further pre-clinical evaluation.