bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 164: β-adrenergic Stimulation Exacerbates Preclinical Arrhythmogenic Right Ventricular Cardiomyopathy Due To Desmoplakin Mutation
Authors: Turdi Subat , Gong Nan , James Jeanne , Winters Patrick , Stanton Lori , Purevjav Enkhsaikhan , Towbin Jeffrey A
Published: July 2021

Background:
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with ventricular arrhythmias and sudden cardiac death. We previously demonstrated that cardiac-specific overexpression of human mutant desmoplakin (DSPR2834H) leads to ARVC in mice at 6 months of age. However, the potential role and mechanism(s) of DSP in preclinical ARVC under cardiac stress remains unclear.

Objectives:
This study is aimed to elucidate the impact of DSP in the development of ARVC under adrenergic stimulation.

Methods:
Three-month-old non-transgenic (NTg), wild-type DSP (Tg-DSPWT) and Tg-DSPR2834H mice without obvious signs of ARVC, including right and left ventricular dysfunction, arrhythmias, were infused with either vehicle or isoproterenol (30mg/kg/d) for 2 weeks using mini-osmotic pumps. During isoproterenol infusion, electrocardiography (ECG) was monitored daily on conscious mice. Echocardiography and cardiac MRI were performed before and after 2-week of isoproterenol infusion. Myocardial tissues from both RV and LV were subjected to cellular, biochemical and histopathological analysis.

Results:
Isoproterenol resulted in cardiac hypertrophy to a similar degree amongst all genotypes; however, mortality occurred only in Tg-DSPR2834H mice. Vehicle-treated mice from all genotypes showed largely normal ECGs, whereas isoproterenol led to various types of arrhythmia in Tg-DSPR2834H mice including ventricular tachycardia and QT prolongation. Echocardiography analysis revealed LV dysfunction (decreased factional shortening) in isoproterenol-treated Tg-DSPR2834H mice compared to other treatment groups. Interstitial fibrosis and lipid infiltration was prominent in the Tg-DSPR2834H myocardium. MRI analysis is being performed to understand the RV and LV geometry and function.

Conclusion:
Our preliminary data confirms the essential role of desmoplakin in response to adrenergic stimulation. Studies investigating the electrophysiological, geometrical and cellular mechanisms of the pro-arrhythmic nature of DSPR2834H dysfunction are ongoing which may ultimately provide critical experimental data on prevention of asymptomatic preclinical ARVC in humans.
Abstract 185: Tripartite Motif E3 Ubiquitin Ligase Family Proteins Regulate Intracellular Signaling During the Progression of Heart Failure
Authors: Alloush Jenna , Beck Eric X , Bhattacharya Sayak , Xu Zhaobin , Guschina Liubov , Manoharan Anjella , Weisleder Noah
Published: July 2021

Heart failure is a major complication of cardiovascular disease that frequently involves initial cardiac hypertrophy that provides transient compensation for decreased heart function. Eventually, decompensation leads to compromised cardiac structure and progression into heart failure. Investigation of the downstream effector pathways for these growth factors has identified molecules involved in the progression of cardiac hypertrophy and heart failure, including phosphoinositide 3-kinase (PI3K) and Akt (Protein Kinase B). MG53, a tripartite motif (TRIM) protein family member designated as TRIM72, is highly expressed in skeletal and cardiac muscle and is known to have cardioprotective effects through modulation of PI3K signaling mechanisms. It is essential for the activation of PI3K-mediated intracellular signaling in cardiomyocytes and TRIM72 overexpression is sufficient to induce PI3K signaling. As TRIM72 regulates PI3K signaling it may play a role in regulation of heart failure, which is supported by our findings that TRIM72 levels increase in the failing mouse heart. Our recent studies also show that TRIM72 can form heterodimers with other members of the TRIM family proteins that contains approximately 70 different members in the human genome. Many TRIM family proteins are known to act as E3 ubiquitin ligases that target the ubiquitin proteasome to particular proteins. Through this activity, TRIM72 homodimers and heterodimers can resolve specific substrates that can modulate aspects of the PI3K/Akt signaling cascade. Our recent studies have resolved multiple binding partners for TRIM72 in the TRIM family that are co-regulated during heart failure. Resolving the target substrates of the heterodimers formed by these various family members and determining their role in regulating PI3K/Akt signaling mechanisms during cardiac hypertrophy will be further defined as our studies continue.
Abstract 186: Identification of Novel Protein Kinase G I Alpha Antiremodeling Substrates in the Myocardium
Authors: Blanton Robert M , Baumgartner Robert A , Thoonen Robrecht , Giovanni Shewit , Govindan Suresh , Aronovitz Mark , Sadayappan Sakthivel , Kass David A , Karas Richard H
Published: July 2021

Protein kinase G I α (PKGIα) inhibits cardiac remodeling, and this effect requires the PKGIα leucine zipper (LZ) binding domain. However, PKGIα LZ-dependent cardiac substrates remain poorly understood. Clinical trials of PKGI activating drugs have been limited to date by hypotension arising from vascular PKGI activation. Therefore, we explored downstream PKGIα substrates in the heart which may inhibit remodeling, yet circumvent the hypotensive effects of systemic PKGI activation. A screen for PKGIα LZ-interacting proteins identified: 1)cardiac myosin binding protein-C (cMyBP-C) and 2) mixed lineage kinase 3 (MLK3).

cMyBP-C is a cardiac myocyte protein known to inhibit remodeling when phosphorylated. Co-precipitations with cGMP-conjugated beads confirmed the PKGIα-cMyBP-C interaction. Purified PKGIα phosphorylated cMyBP-C in vitro at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these sites in COS cells transfected with WT PKGIα, but not in cells transfected with either LZ mutant PKGIα or kinase-inactive PKGIα. In hearts of 9 month old PKGIα Leucine Zipper mutant mice, which have LV hypertrophy (LVH) and diastolic dysfunction, we observed decreased phosphorylated cMyBP-C as well as decreased total cMyBP-C, compared with WT littermate hearts.

We next tested the effect of MLK3, which interacts with PKGIα in the heart, on remodeling in vivo. We performed 7 day Transaortic Constriction (TAC) on MLK3 KO mice and WT littermates (n=5 shams, 8 TAC per genotype). MLK3 KO TAC mice had increased LVH (LV mass/tibia length 71.1 ± 2.7 g/cm KO TAC vs 62.1 ± 2.7 WT TAC; p<0.05). Further, MLK3 KO mice developed overt CHF compared with WT littermates (LV end diastolic pressure 14.8 ± 1.9 mmHg KO TAC vs 7.7 ± 2.1 WT TAC, p <0.05), as well as accelerated decrements in LV preload recruitable stroke work (36.6 ± 11.9 mmHg/ul KO TAC vs 94.6 ± 12.9 WT TAC, p<0.05) and min dP/dt (-6292 ± 519 mmHg/s KO TAC vs −8157 ± 554 WT TAC , p <0.05). We observed no differences in LV structure or function between sham genotypes.

These studies reveal 2 novel PKGIα anti-remodeling substrates, and they support that exploring PKGIα substrates in the heart may identify novel therapeutic targets to inhibit cardiac remodeling but avoid excessive PKGI induced vasodilation.
Abstract 187: αB-crystallin Interacts And Prevents Stress-activated Proteolysis Of Focal Adhesion Kinase In Cardiomyocytes
Authors: dos Santos Aline M , Pereira Michelle B , Gonçalves Danieli C , Cardoso Alisson C , Consonni Silvio R , Gozzo Fabio C , Oliveira Paulo S , Figueiredo Alana R , de Thomaz André A , Cesar Carlos L , Ramos Carlos H , Franchini Kleber G
Published: July 2021

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here, we show that αB-crystallin confers protection to FAK against calpain-mediated proteolysis under mechanical stress in cardiomyocytes. Biochemical assays, chemical cross-linking coupled to mass spectrometry experiments, mutational analyses and Förster resonance energy transfer (FRET) were combined to investigate the basis of FAK and αB-crystallin interaction. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation by mechanical stress. αB-crystallin silencing results in calpain-dependent FAK depletion and in increased apoptosis of cardiomyocytes. The overexpression of a myc-FAK construct or treatment with a calpain inhibitor (E64) restored the survival of cardiomyocytes depleted of αB-crystallin. The association between FAK and αB-crystallin was also demonstrated to occur in response to pressure overload in rat left ventricle. The myocardial depletion of αB-crystallin by siRNA results in enhanced apoptosis of cardiomyocytes and myocardial fibrosis in overloaded hearts. These alterations were markedly attenuated in the overloaded left ventricles of transgenic mice with cardiac specific FAK expression. These findings define a novel mechanism by which αB-crystallin controls FAK function, with impact on cardiomyocyte survival and cardiac remodelling in response to stress.
Abstract 19: Reversal of Adverse Remodeling in Chronic Rat Myocardial Infarction by Cardiospheres: Role of the Tgf-beta Pathway
Authors: TSELIOU ELENI , de Couto Geoffrey , Terrovitis John , Sun Baiming , Liu Weixin , Middleton Ryan , Marbán Linda , Marbán Eduardo
Published: July 2021

Background:
Self-assembling three-dimensional cardiospheres (CSps) are known to be effective in improving function and attenuating remodeling as adjunctive therapy in acute myocardial infarction (MI), and they exert functional benefits in large animals with convalescent MI. The effect of cardiospheres in chronically-remodeled myocardium post-MI, and the mechanisms of benefit are unknown.

Methods:
We performed permanent LAD ligation in Wistar Kyoto female rats. One month later the animals underwent repeat thoracotomy and were randomly assigned to one of two groups: vehicle-treated controls, or CSp-treated, where CSps (at a seeding dose of 2X10
6
cells) were injected in the peri-infarct area. Mechanisms were also probed
in vitro
.

Results:
CSps downregulated the canonical Tgfβ1-smad2/3 pathway in co-cultures with fibroblasts and reduced fibroblast proliferation
in vitro
. Soluble endoglin, abundantly expressed in CSps, was found to limit the Tgfβ1-induced fibrosis in a dose-dependent manner.
In vivo
, left ventricular function gradually increased one month post-CSp injection with further improvement after up to six months of follow up. Immunoblots verified Tgfβ1/smad cascade downregulation one month post-CSp injection and upregulation of soluble endoglin. In addition, scar mass was reduced one month post-CSp injection compared to controls. Collagen density was attenuated within the infarcted region of the treated group, while immunostaining showed enhanced vessel density in the same region. Six months post-transplantation, cardiomyocyte hypertrophy was reduced in the CSp-treated group compared to control.

Conclusions:
CSps injected into the peri-infarct zone enhance angiogenesis and reduce established fibrosis in chronically-infarcted myocardium, thereby reversing, at least partially, adverse remodeling of the heart. The underlying mechanism involves modification of Tgfβ1/smad signaling, a key pathway in the cardiac response to injury.
Abstract 192: Scleraxis is a Required Component of the Cardiac Extracellular Matrix Gene Expression Program
Authors: Czubryt Michael P , Bagchi Rushita A , Roche Patricia L , Wang Ryan , Devalapurkar Shreyas , Schweitzer Ronen
Published: July 2021

The collagenous cardiac extracellular matrix (ECM) reinforces the myocardium and facilitates intercellular communication, but excessive ECM production in fibrosis results in dysfunction and heart failure. The transcription factor scleraxis directly governs expression of type I fibrillar collagen in ECM-rich tissues including tendons and the heart. We have also shown that scleraxis is required for collagen 1α2 gene expression induced by the potent pro-fibrotic TGFβ-Smad signaling pathway. We therefore examined the broader role of scleraxis in myocardial ECM production, including mechanisms regulating the expression of scleraxis itself. Scleraxis knockout in mice resulted in a dramatic ~50% loss of cardiac ECM components including fibrillar collagens, proteoglycans and matrix metalloproteinases, concomitant with a significant decrease in fibroblast number. Scleraxis knockdown in primary cardiac proto-myofibroblasts recapitulated these changes without increasing cell death, suggesting a reduction in fibroblast precursors in knockout mice in vivo. Conversely, over-expression of scleraxis had the opposite effect, up-regulating expression of ECM genes and numerous markers indicative of increased commitment to a myofibroblast cell fate. Scleraxis increased proto-myofibroblast contractility via direct transactivation of the α-smooth muscle actin promoter. Similar to TGFβ, pro-fibrotic angiotensin II and Connective Tissue Growth Factor induced scleraxis expression, suggesting that scleraxis behaves as a common transcriptional effector for multiple fibrotic pathways. Fibrillar collagen gene expression induced by these factors was significantly attenuated by scleraxis knockdown, further implicating scleraxis in fibrotic ECM synthesis. Intriguingly, the histone deacetylase inhibitor trichostatin A, which has been reported to exert anti-fibrotic effects in the heart, significantly reduced scleraxis expression in cardiac myofibroblasts. These data collectively identify scleraxis as a central and requisite transcriptional regulator of fibroblast phenotype and the ECM gene expression program in the heart, and provide rationale for the investigation of anti-scleraxis strategies to attenuate fibrosis in patients.
Abstract 199: Specific MicroRNAs Regulate Cardiac Fibroblast-to-Myofibroblast Transition And Fibrosis.
Authors: Nagpal Varun , Rai Rahul , Place Aaron , Murphy Sheila , Vaughan Douglas E
Published: July 2021

Cardiac fibrosis is the pathological consequence of fibroblast-to-myofibroblast transition (FMT) within the human myocardium, resulting in heart dysfunction. Transforming growth factor-β (TGF-β) plays a pivotal role in the induction of both FMT and cardiac fibrosis. However, the molecular basis of TGF-β-induced FMT in cardiac fibrosis is not clear. In this study, we propose a novel miRNA-mediated approach to attenuate cardiac fibrosis by blocking TGF-β-induced FMT. We observed that the canonical TGF-β/SMAD pathway, and not the MEK pathway, plays a pivotal role in the induction of FMT in primary cultures of human cardiac fibroblasts. Importantly, we have demonstrated that the specific miRNA is significantly upregulated during cardiac FMT. In addition, we observed significant upregulation of the same miRNA in fibrotic human myocardium and two murine models of cardiac fibrosis (transverse aortic constriction and Angiotensin II). Furthermore, overexpression of the miRNA using mimics augmented TGF-β-induced FMT. Downregulation of the miRNA using an antagomiR approach attenuated TGF-β-induced FMT. Notably, in silico analysis and qRT-PCR analysis revealed that this miRNA directly targets apelin, an anti-fibrotic mediator. Next, efficient delivery of cy3-tagged antagomiRs in the heart, liver and spleen was confirmed by confocal microscopy. In vivo silencing of miRNAs in the heart was achieved by systemic delivery of locked nucleic acid (LNA), both in the presence and absence of Angiotensin II. We conclude that TGF-β-induced specific miRNA is both sufficient and necessary for the induction of cardiac FMT and is a novel repressor of apelin. Our data suggests that the inhibition of miRNAs necessary for FMT may serve as a novel therapeutic strategy to prevent human cardiac fibrosis.
Abstract 212: Prophylactic Anti-inflammatory Treatment Attenuates The Neuroinflammatory And Behavioral Effects Of Silent Cerebral Infarction
Authors: Nemeth Christina L , Neigh Gretchen N
Published: July 2021

Silent brain infarction is a frequent complication of cardiac surgery and is associated with mood changes and cognitive disruption. Microsphere embolism (ME) rodent models recapitulate both the diffuse ischemic infarcts and the delayed subtle behavioral disturbances characteristic to silent infarction (SI). Previously, we have shown that ME leads to increased hippocampal inflammation, weakening of the blood brain barrier, and the infiltration of peripherally circulating inflammatory cells in rats. Given long-term increases in inflammatory activity following SI, the current study tests the efficacy of anti-inflammatory versus anti-depressant treatment strategies to reduce the inflammatory and behavioral sequelae of injury. Adult rats were administered either chronic meloxicam (preferential COX-2 inhibitor) or fluoxetine (SSRI) beginning five days prior to ME surgeries. After a two week recovery, animals were tested for anxiety-like behaviors in the open field paradigm and the hippocampus was examined for gene expression of inflammatory cytokines. Meloxicam treated animals showed a decrease in hippocampal gene expression of inflammatory markers (SPP1; p = 0.0272) and greater than a 3-fold change improvement in open field central tendency (p = 0.0003). No differences in inflammatory gene expression were observed in fluoxetine treated animals (SPP1; p = 0.3288); however, fluoxetine treatment resulted in a 2-fold change improvement in open field central tendency (p = 0.0138) suggesting that while both treatment strategies attenuate SI induced behavioral disruption, only meloxicam acts via inflammatory mechanisms. Given the long term negative consequences of increased central and peripheral inflammatory activity, the data suggest that anti-inflammatory therapeutic strategies may benefit patients at risk for SI as well as cardiac surgery candidates.
Abstract 214: Reduction of Scar Tissue after GHRH-A Treatment in a Swine Model of Sub-acute Ischemic Cardiomyopathy
Authors: Bagno Luiza L , Kanashiro Takeuchi Rosemeire , Wang Bo , Suncion Viky , Karantalis Vasileios , Premer Courtney , Golpanian Samuel , Balkan Wayne , Rodriguez José E , Valdes David , Rosado Marcos , Wolf Ariel , Block Norman L , Goldstein Peter , Morales Azorides , Schally Andrew V , Hare Joshua M
Published: July 2021

Background:
Growth hormone-releasing hormone receptor agonists (GHRH-A) stimulate cardiac repair following myocardial infarction (MI) through the activation of the GHRH signaling pathway within the heart. We tested the hypothesis that the administration of GHRH-A prevents ventricular remodeling in a swine sub-acute MI model.

Methods:
Twelve female Yorkshire swine (25-30 Kg) underwent transient occlusion of the LAD coronary artery (MI). Two-weeks post-MI, swine were randomized to receive injections of either 30 μg/Kg GHRH-A (MR-409) (GHRH-A group; n=6) or vehicle (placebo group; n=6). Cardiac MRI, pressure volume loops and measures of endothelial function were obtained at multiple time points. Infarct-, border- and remote- (non-infarcted) zones were assessed by immunohistochemistry for the growth hormone-releasing hormone receptor (GHRHR).

Results:
Four-weeks of GHRH-A treatment resulted in reduced scar mass (GHRH-A group: –21.9±6.42%; p=0.02; placebo group: 10.9±5.88%; p=0.25; Two-way ANOVA; p=0.003), and reduced scar size (percent of left ventricle mass) (GHRH-A group: –38.38±4.63; p=0.0002; placebo group: –14.56± 6.92; p=0.16; Two-way ANOVA; p=0.02). Moreover, peripheral endothelial function was significantly increased compared to baseline values in the GHRH-A group (paired t-test; p=0.006) but not in the placebo group (p=0.99). Unlike in rats, this reduced infarct size in swine was not accompanied by improved cardiac function as measured by serial hemodynamic pressure-volume analysis. GHRH receptors were abundant in cardiac tissue, with a greater density in the border zone of the GHRH-A group compared to the placebo group. These data support the concept of direct post-infarction activation of cardiac signal transduction, and of enhancing this activation with systemic treatment by GHRH.

Conclusions:
Daily subcutaneous administration of GHRH-A is feasible and safe in female swine. Furthermore, GHRH-A therapy significantly reduced infarct size and increased endothelial function, suggesting that a local activation of the GHRH pathway leads to the regenerative process and preservation of peripheral endothelial function.
Abstract 227: Activation of Angiotensin II Type 2 Receptor Suppresses TNFα-induced ICAM-1 via NF-κB: Possible Role of ACE2
Authors: Zhu Liping , Carretero Oscar A , Xu Jiang , Harding Pamela , Yang Xiao-Ping
Published: July 2021

Activation of the renin-angiotensin system (RAS) is a major factor contributing to the pathophysiology of cardiovascular disease (CVD). Blockade of the RAS with angiotensin receptor blockers (ARBs) has been a standard treatment for CVD. Activation of angiotensin II type 2 receptor (AT2) and angiotensin I-converting enzyme 2 (ACE2) contribute to the cardioprotective effects of ARBs. Both AT2 and ACE2 counteract the vasoconstrictor and pro-inflammatory effects of angiotensin II. However, the possible interaction between AT2 and ACE2 has never been established. Tumor necrosis factor (TNFα) is a cytokine involved in angiotensin II signaling and promotes the inflammatory response via NF-κB. We hypothesized that activation of AT2 increases ACE2, thereby preventing TNFα-stimulated intercellular adhesion molecule-1 (ICAM-1) expression via inhibition of NF-κB. Human coronary artery endothelial cells were pretreated with AT2 antagonist PD123319 or ACE2 inhibitor DX-600, and then stimulated with TNFα in the presence or absence of AT2 agonist CGP42112A. ACE2 mRNA was measured by real-time RT-PCR. ACE2 activity was measured using a Fluorimetric Kit. ICAM-1 and phospho-inhibitory κB (p-IκB) were measured by Western Blot. Activation of AT2 with CGP42112A increased ACE2 mRNA by 1.82 ± 0.09 fold (p<0.01) and ACE2 activity from 0.61 ± 0.05 to 0.95 ± 0.03 (pg/μl/h/μg protein) (p<0.01). This effect was diminished by inhibition of AT2 or ACE2. ICAM-1 expression was almost undetectable in untreated cells but greatly increased by TNFα. Activation of AT2 reduced TNFα-induced ICAM-1 expression by 47% (from control value of 1 to 0.53 units) ± 5% (p<0.01), which was diminished by AT2 antagonist or ACE2 inhibitor. We also found that TNFα increased p-IκB by 7.46 ± 0.51 fold (p<0.01) compared to untreated cells and this effect was diminished by AT2 activation. Furthermore, ACE2 inhibitor blunted the effects of AT2 on TNFα-induced p-IκB expression. Our findings suggest that stimulation of AT2 reduces TNFα-induced ICAM-1 expression, which is partly through ACE2-mediated inhibition of NF-κB. Perspective: understanding the mechanisms underlying AT2-mediated anti-inflammatory effects could lead to new therapeutic strategies such as specific activation of AT2 and/or ACE2.
Abstract 235: Molecular Signature Of Cardiomyogenic c-kit-positive Bone Marrow Cells
Authors: Kim Junghyun , Sanada Fumihiro , Pereira João D , Czarna Anna , Churko Jared , Wu Joseph C , Rota Marcello , Anversa Piero , Leri Annarosa
Published: July 2021

Conflicting results have been reported concerning the ability of c-kit-positive bone marrow cells (c-kit-BMCs) to transdifferentiate into cardiomyocytes in vivo. We have raised the possibility that c-kit-BMCs may constitute a functionally heterogeneous pool, containing cells with different cardiomyogenic potential. To test this hypothesis, clonal analysis of individual c-kit-BMCs was introduced to track in vivo the progeny derived from a single founder cell. By employing viral gene-tagging, only a limited fraction of clonal c-kit-BMC was found to be able to generate cardiomyocytes in the acutely infarcted heart. On this basis, c-kit-BMC-derived clones were classified as myogenic or non-myogenic. The gene profile of these two categories of cells was studied by RNA-sequencing to determine whether myogenic c-kit-BMCs had a distinct molecular signature. Genes that showed a significant (p<0.05) expression difference of at least 2-fold among cell groups were included in the analysis; 1243 genes were upregulated and 493 genes were downregulated in myogenic c-kit-BMCs when compared with non-myogenic c-kit-BMCs. An additional comparison involved freshly isolated c-kit-BMCs. 2512 genes were upregulated in myogenic c-kit-BMCs and 1844 genes were downregulated in this cell group. These results were subjected to functional annotation using gene ontology for the identification of molecular pathways showing different state of activation or repression in myogenic BMCs. The affected transcripts in myogenic c-kit-BMCs involved mostly signal transduction and muscle development including cardiac muscle. Similarly, a set of genes implicated in the regulation of angiogenesis were more represented in myogenic c-kit-BMCs, possibly reflecting the proficiency of this cell class to form vessels in the microenvironment of the infarcted heart. Our findings document that myogenic clonal c-kit-BMCs have a distinct molecular signature that differs from that of non-moygenic and non-expanded c-kit-BMCs. On this basis, c-kit-BMCs with high cardiomyogenic ability can be prospectively isolated and employed clinically for the treatment of patients with cardiac diseases.
Abstract 246: Translocation Of Myomirs Via Gap Junction Channels Prevents Cancer Cell Growth
Authors: Graciotti Laura , Hosoda Toru , Rota Marcello , Borghetti Giulia , Signore Sergio , Anversa Piero , Leri Annarosa
Published: July 2021

The adult heart is resistant to cancer formation and the metastatic invasion of distant neoplasms. This biological advantage may be dictated by the molecular properties of myocytes that constitutes 90% of the myocardium. We raised the possibility that microRNAs (miRs) highly expressed in myocytes (myomirs) may translocate via gap junctions to neighboring cancer cells, preventing their growth or inhibiting their survival. First, we established whether overexpression of myomirs interferes with the proliferation and death of MCF7 human breast cancer cells. Infection of MCF7 with lentiviruses carrying miR-1, miR-133a and miR-499 (miR-MCF7) resulted in a 5-fold decrease in Ki67 labeling and a 20% increase in the fraction of cells arrested at G0/G1. In contrast, TdT-positive apoptotic cells averaged 0.5% and did not differ in miR-MCF7 and control cells. To mimic the in vivo condition, EGFP-labeled MCF7 were co-cultured with myocytes and, 4 days later, the expression of myomirs was measured in FACS-sorted MCF7. With respect to baseline, co-cultured MCF7 showed 100-fold, 16-fold, and 27-fold increase in the expression of miR-1, miR-133a and miR-499, respectively. Co-culture of myocytes and MCF7 led to the formation of gap junctions made of connexin 43 (Cx43) and connexin 45 (Cx45). Silencing of Cx43 and Cx45 decreased significantly the expression of myomirs in co-cultured MCF7. Importantly, proximity of MCF7 to myocytes reduced markedly the growth rate of the cancer cells. Subsequently, 1 x 106 MCF7 or miR-MCF7 were injected subcutaneously in NOD-scid mice. At 5 weeks, the tumors developed from miR-MCF7 were 70% smaller than those originated from control MCF7. Two doses of breast cancer cells were injected intramyocardially to establish their in situ tumorigenic effects. Tumor formation was found in all hearts that received 1 x 106 MCF7. Conversely, mice injected with 1 x 105 cells did not show macroscopic evidence of neoplastic lesions. The lack of tumor development in the latter case is consistent with the ability of the heart to prevent neoplasm development when cancer cell colonization is not massive. Our findings document that miR-1, miR-133a and miR-499 translocate from myocytes to cancer cells via gap junctions, inhibiting tumor growth in vitro and in vivo.
Abstract 249: Mir-211 Regulates Bone Marrow Mesenchymal Stem Cells Migration Through Stat5a
Authors: Hu Xin Yang , Chen Panpan , Wu Yan , Zhu Wei , Wang Jian-an
Published: July 2021

Background:
Efficacy of intravenous mesenchymal stem cells (MSCs) administration for myocardial infarction (MI) is limited by low cell migration to the damaged myocardium. Our previous study demostrated that migration ability of MSCs enhanced by hypoxia preconditioning (HPC). miRNA microarray displayed that miR-211 exhibited most significant change between HPC and normoxia cultured MSCs. The aim of this study is to study whether and how miR-211 regulate MSCs migration.

Methods:
In vitro, transwell assay were used to assess the migration ability of MSC moduated by miR-211 using overexpressing and knockdown lentivirus. The target gene of miR-211 predicted by Targetscan were verified by PCR, western blot and lucifurase assay. Chromatin immunoprecitation (ChiP) were used to explore the transcription factors that regulate the expression of miR-211. To evaluate the effect of miR-211 on MSCs migration in vivo, miR-211-mimic and miR-211-shRNA male MSCs were intravenously delivered 24h after MI, the engraft cells were detected by RT-PCR of SRY gene.

Results:
Quatitative RT-PCR showed that miR-211 expression of MSCs upregulated by HPC. MiR-211 mimic improved MSCs migration by 31.03% (p<0.05), however, knockdown miR-211 using shRNA attenuated MSCs migration ability significantly. Signal transducer and activator of transcription 5A (STAT5A) was predicted as one of miR-211 target genes, PCR and Western blot showed miR-211 overexpression dramatically decreased STAT5A expression, while miR-211 knockdown upregulated STAT5A. The luciferase assay showed the similar results. Transwell assay showed that STAT5A knockdown reverse the inhibition of MSCs migration induced by miR-211-shRNA. Intrestingly, ChiP assay showed that STAT5A can combine to the promoter of miR-211, which lead to the regulation of miR-211 transcription. In vivo data showed that MiR-211 overexpression enhanced MSCs homing to ischemic myocardium, and miR-211 overexprssing MSCs improved cardiac function 28days post-MI. However, miR-211 knockdown decreased MSCs homing and hampered cardiac function recovery.

Conclusions:
These results indicate that miR-211 has important role in regulating MSCs migration through targeting STAT5A, meanwhile STAT5A regulated miR-211 transcription.
Abstract 25: The Role of cAMP-Phosphodiesterase 1C Signaling in Pathological Cardiac Remodeling and Dysfunction
Authors: Knight Walter E , Oikawa Masayoshi , Miller Clint , Yan Chen
Published: July 2021

The cyclic nucleotides cAMP and cGMP play important roles in mediating both protective and detrimental signaling in heart failure. By acting as regulators of cAMP and cGMP, the phosphodiesterases play important roles in modulating this signaling. A comparison of PDE expression between control mice and mice given transverse aortic constriction revealed that expression of phosphodiesterase 1C (PDE1C) was increased significantly by TAC, both on the mRNA and protein levels. To determine whether this was protective or maladaptive, we performed TAC on mice with a genetic deletion of PDE1C. While TAC-operated WT mice experienced significant overall cardiac hypertrophy and cardiomyocyte hypertrophy, these were reduced in PDE1C KO mice. Cardiac function, as assessed by echocardiography, was also reduced significantly in WT TAC mice, but was preserved in PDE1C KO TAC mice. Histological analysis indicated that TAC-operated PDE1C KO mice also experienced reduced cardiomyocyte apoptosis compared to WT mice, indicating a potential cardioprotective mechanism for PDE1C deletion. Cardiomyocytes isolated from PDE1C KO mice experienced reduced Ang II or Iso-induced cell death compared to WT myocytes, indicating that this was a cardiomyocytes-specific effect of PDE1C deletion. Ang II-induced cardiomyocyte cell death and apoptosis were also blocked via pharmacological PDE1 inhibition. PDE1C is able to hydrolyze either cAMP or cGMP; therefore, it seemed possible that this protective mechanism was dependent on either PKA- or PKG-mediated signaling. While PKG inhibition did not alter the protective effect of PDE1 inhibition in isolated cardiomyocytes, PKA inhibition blocked it. Overexpression studies also indicated that PDE1C is localized to the cell membrane in cardiomyocytes. Therefore, we propose that by modulating a novel, membrane-localized, anti-apoptotic, cAMP/PKA-dependent pathway in cardiomyocytes, PDE1C potentially represents a novel therapeutic target in heart failure.
Abstract 268: Transforming Growth Factor-β1 Increases Resistance of Fibroblasts to Apoptotic Cell Death
Authors: Negmadjanov Ulugbek , Godic Zarko , Mirza Mahek , Emelyanova Larisa , Rizvi Farhan , Holmuhamedov Ekhson , Jahangir Arshad
Published: July 2021

Introduction:
Cardiac injury results in the death of cardiac myocytes and subsequent scar formation through extracellular matrix (ECM) deposition by fibroblasts (FB) and myofibroblasts (myoFB). Excessive fibrosis results in pathological scarring that predisposes to arrhythmogenesis and heart failure, particularly in the elderly. Strategies to limit adverse ECM remodeling are urgently needed to curtail the growing epidemic of atrial fibrillation and heart failure in the aging population. Persistence of myoFB and resistance to apoptotic cell death has been proposed to underlie the mechanism of excessive fibrosis, yet is not fully characterized.

Methods:
Cultured NIH/3T3 cells (control and TGF-β1 treated) have been challenged with activators of extrinsic (FAS-Ligand, 1 μg/mL) or intrinsic (Thapsigargin 10 μM and Staurosporine 5 μM) apoptotic pathways and Caspase-3 activity was measured in cellular lysate.

Results:
FAS-L exposure induced ~40-fold suppression of Caspase-3 activity in TGF-β1 treated cells as compared with control (17±12 vs 686±5 nmol AMC/min/106 cells, respectively). Similarly, Staurosporine activated Caspase-3 in TGF-β1 treated cells ~3-fold (171±38 vs 536±29 nmol AMC/min/106 cells), and Thapsigargin ~10-fold (73±33 vs 742±8 nmol AMC/min/106 cells).

Conclusion:
TGF-β1 treatment increased the sensitivity of NIH/3T3 cells toward extrinsic and intrinsic apoptotic stimuli. Although, TGF-β1 treatment increased overall resistance of NIH/3T3 cells to apoptosis, the responsiveness of cells to extrinsic vs intrinsic pathways was differentially affected. This data support the hypothesis that persistence of myoFB results in pathological scarring.
Abstract 275: Electrophysiological Consequences of AAV9 mediated SERCA2a Gene Transfer to Normal Rat Myocardium
Authors: Xie Chaoqin , Jeong Dongtak , Kohlbrenner Erik , Hajjar Roger J , Akar Fadi G
Published: July 2021

SERCA2a gene transfer to the failing heart improves its mechanical function and inhibits arrhythmic triggers caused by aberrant calcium waves. The majority of arrhythmias occur before onset of end-stage heart failure when SR function is not compromised. Examination of the electrophysiological (EP) consequences of SERCA2a gene therapy in normal hearts is imperative in determining its potential toxicity as a preventive therapy during early stages of remodeling. We hypothesized that AAV9 mediated gene transfer of SERCA2a to normal rat hearts may promote the incidence of arrhythmic triggers under conditions of intracellular calcium (Ca2+) overload.

Methods:
Rats underwent tail vein injection of 5x10E11vg AAV9.SERCA2a (N=8) or AAV9.GFP (N=3). 6 naïve uninjected rats served as controls (Ctl). Using high resolution optical action potential (AP) imaging, we characterized the EP substrate 6 weeks post gene transfer. Ex vivo hearts were examined during normoxic perfusion and challenge with low flow ischemia for 1h followed by reperfusion. Arrhythmia susceptibility was determined under conditions of Ca2+ overload.

Results:
Surprisingly, AAV9.GFP hearts exhibited markedly prolonged AP durations (APD) and slower conduction velocities (CV) compared to AAV9.SERCA2a and Ctl hearts. Challenge of AAV9.GFP but not AAV9.SERCA2a or Ctl hearts with burst pacing always resulted in VF. Remarkably, APD & CV rate relationships were identical in Ctl and AAV9.SERCA2a hearts (p=NS). Elevation of pacing rate resulted in a comparable level of AP alternans followed by loss of 1:1 capture in both groups. No evidence of triggered APs was found in AAV9.SERCA2a or Ctl hearts in response to burst stimulation. Ischemia for 1hr produced comparable changes in APD and CV. Challenge with burst stimuli during ischemia elicited arrhythmias in 2/6 Ctl and 6/8 AAV9.SERCA2a hearts (p=0.277, NS). Upon reperfusion, 1/4 & 0/2 remaining Ctl and AAV9.SERCA2a hearts exhibited VF.

Conclusion:
Surprisingly, AAV9.GFP but not AAV9.SERCA2a gene transfer to normal rat myocardium alters EP properties and promotes Ca2+ mediated arrhythmias. Lack of an EP phenotype of AAV9.SERCA2a suggests an upper limit of endogenous SERCA2a expression in normal rodent myocardium that is not exceeded by gene transfer.
Abstract 278: Diet-Induced Leukocyte Telomere Shortening Correlates with Extent of Atherosclerosis
Authors: KARERE GENESIO , Birnbaum Shifra , Christensen Clint , Mahaney Michael , VandeBerg John , Cox Laura
Published: July 2021

Introduction:
Cardiovascular disease, the leading cause of death in developed countries, is commonly due to atherosclerosis. Studies have demonstrated association between leukocyte telomere shortening (LTS), extent of atherosclerotic lesions and accelerated cell senescence. Further LTS is associated with dietary intake. However, efforts to link LTS, diet and extent of lesions have been unsuccessful in humans due to difficulties controlling diet in large human population studies. To begin addressing these critical issues, we controlled dietary fat (high-fat, HF) in baboons for 2yrs - a well-developed primate model of human atherosclerosis. This is the first study in primates showing correlation of LTS with both chronic HF diet and atherosclerotic lesions.

Hypothesis:
We hypothesized that leukocyte telomere length decreased with chronic HF diet in baboons and is correlated with extent of atherosclerotic lesions.

Methods and Results:
A cohort of pedigreed baboons (n=107; females=46, males=61) was fed a HF diet for 2yrs. Absolute leukocyte telomere lengths (LTL; kb/diploid genome) were quantified by qPCR before and after diet challenge. Total telomere length was calculated by computing the ratio of telomere quantity per single copy gene quantity (baboon LIPG). Mean LTL was significantly shorter after feeding baboons a HF diet for 2 yrs (paired t test, p=0.03). Baboons (n=232) maintained on a low fat diet for 2yrs showed no significant difference in LTL (p=0.47). These findings suggest that a HF diet accelerates LTS. Further we quantified the extent of atherosclerotic lesions in baboons after 2yr HF diet and found that LTL, adjusted for age and sex, were correlated with lesions in descending aorta (Pearson correlation, r=0.19; p=0.03). Interestingly this correlation was significant in females but not in males after adjusting for age (r=0.27, p=0.03).

Conclusions:
LTS correlates with chronic feeding with a HF diet in baboons, is significantly correlated with arterial lesions and the correlation is sex-specific. These findings suggest that LTS may be a potential biomarker of extent of atherosclerosis.
Abstract 280: Thrombospondin-1 Carries CD55 like Domains: A Novel Target for E. coli Invasion into Human Heart Cells
Authors: Rana Tanu , Nde Pius N , Nowicki Stella , Udoko Aniekanabasi N , Singh Rajbir , Johnson Candice A , Dykan Andrey , Lawler Jack , Villata Fernando , Nowicki Bogdan J
Published: July 2021

Introduction:
E. coli septicemia with cardiovascular dysfunction is among most frequent causes of human mortality however, underlying molecular mechanisms remain unknown. We recently described a fatal case of uro-sepsis with E. coli, bearing invasion-ligand Dr-adhesin, resulting in myocardial necrosis. Here we investigated an unrecognized mechanism of pathogen mediated cardiac cell invasion as a contributing factor to cardiovascular pathology and patient mortality.

Methods:
The experiments on human cardiac fibroblasts (HCF) showed E. coli colonization and invasion. However, the primary cell receptor for Dr adhesin, CD55, was undetectable. Searching for an alternative receptor we focused on matrix protein TSP-1 which is elevated in heart during infection. We over-expressed TSP1 in HCF cells (HCF-TSP1) and challenged with Dr+ E. coli. Bacterial binding/invasion to HCF-TSP1 and wild-type (WT) HCF were compared and evaluated. To rule out the possibility of fibroblast invasion due to presence of CD55 we performed Western blotting with CD55 antibody. Analysis of TSP-1 and CD55 epitopes was done to look for bacteria binding domains.

Results:
A dose-dependent TSP-1-E. coli binding assay in triplicates showed bacteria-HCF specific interactions. HCF-TSP1 had 13±4 bacteria bound per cell and invasion level of 2000/well which was up to 2.5 fold higher compared to WT-HCF. Both immunostaining and immunoblotting showed presence of TSP1 and absence of CD55 in HCF-TSP1. The interaction between purified/recombinant TSP1 coated to microtiter plates showed dose-dependent attachment of E. coli which increased with TSP1 concentrations. Sequence analysis of TSP-1 and CD55-SCR-3 revealed multiple 12AA CD55-like sequences within TSP-1. CD55-like epitopes identified on TSP1 shared up to 80% identity with the CD55 functional epitopes required for complement inhibition, Dr+ E. coli binding and invasion.

Conclusions:
Here we identified cardiac fibroblasts as target for uro-septic E. coli invasion. We discovered TSP-1 as the receptor facilitating Dr+ E. coli invasion process and identified previously unrecognized CD55-like sequences within TSP-1 which in CD55 account for functional epitopes allowing E. coli attachment, invasion and complement inhibition.
Abstract 284: Cyclic stretch of Embryonic Cardiomyocytes Increases Proliferation, Growth, and Expression While Repressing Tgf-β Signaling
Authors: Banerjee Indroneal , Carrion Katrina , Serrano Ricardo , Dyo Jeffrey , Lund Sean , Willems Erik , Aceves Seema , Meili Rudolph , Mercola Mark , Chen Ju , Zambon Alexander , Zambon Alexander , Hardiman Gary , Doherty Taylor A , del Álamo Juan C , Nigam Vishal
Published: July 2021

Rationale:
Perturbed biomechanical stimuli are critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While ventricular cardiomyocytes experience biomechanical stretch every heart beat, the molecular responses of embryonic cardiomyocytes to biomechanical stimuli are poorly understood. In this study, we examined how cyclic mechanical stretch modulates embryonic cardiomyocytes to improve understanding of normal and pathologic ventricular development. We hypothesize that biomechanical stimuli activates specific signaling pathways that impact proliferation, gene expression and myocyte contraction.

Objective:
The objective for this study was to examine key molecular and phenotypic responses of embryonic cardiomyocytes to cyclic stretch that will provide a deeper understanding of HLHS.

Methods and Results:
Embryonic mouse cardiomyocytes were exposed to cyclic stretch. Analysis of RNA-Sequencing data demonstrated that gene ontology (GO) groups associated with myofibril and cardiac development were significantly modulated. Stretch increased cardiomyocyte proliferation, size, and cardiac gene expression. Since the Tgf-β GO term was modulated by stretch, the role of Tgf-β in the cardiomyocyte response to stretch was examined. Stretched Cardiomyocytes had decreased Tgf-β expression, protein, and signaling. Functionally, Tgf-β signaling repressed cardiomyocyte proliferation, and both inotropic and chronotropic contractile function, which was assayed for confluent cell cultures by dynamic monolayer force microscopy (DMFM). Tgf-β inhibitor treatment resulted in increased cardiomyocyte size.

Conclusions:
Herein, we observed that cyclic stretch promotes cardiomyocyte proliferation, growth, and gene expression. Stretch-mediated repression of Tgf-β appears to play a key role. Together these findings advance the understanding of how the biomechanical/molecular axis modulates ventricular development.
Abstract 289: Retinoblastoma Gene Deletion Promotes Cardiac Dysfunction, Fibrosis and Apoptosis in Response to Pressure Overload
Authors: Ammann Paul B , Yamamoto Takanobu , Zhai Peiyong , Sadoshima Junichi
Published: July 2021

The retinoblastoma (Rb) protein is a universal cell cycle regulator in mammals. When the Rb protein is phosphorylated by Cyclins/Cdks, it dissociates from E2F, and Rb-dependent E2F repression is subsequently inactivated. Furthermore, the Rb protein has also been implicated in the regulation of cardiac hypertrophy and apoptosis in cardiomyocytes (CMs).

To elucidate the role of Rb in response to mechanical stress, we conducted transverse aortic constriction (TAC) in cardiac-specific Rb knockout mice (cRb-KO) in vivo (C57BL/6J). Cardiac-specific deletion of Rb was achieved by crossing Rb flox/flox mice with αMHC-Cre mice.

Under basal conditions, 3- to 5-month-old cRb-KO mice showed increased heart weight (HW) (left ventricular weight/ tibial length (TL): 5.93 ± 029 vs. 4.76 ± 0.14, p< 0.01), increased apoptosis as determined by TUNEL staining (0.12% vs. 0.02%, p< 0.05) and a trend towards cardiac dysfunction (-dP/dt: 4320 ± 388 vs. 5933 ± 489 mmHg/sec, p < 0.05) compared to control mice (Rb flox/flox)

Following 2 weeks of TAC, cRb-KO mice showed increased heart weight (HW/TL: 8.58 ± 0.35 vs. 7.50 ± 0.24, p < 0.05), cardiac dysfunction (ejection fraction (EF): 51.1% ± 4.0 vs. 74.3% ± 0.9, p < 0.01) , increased apoptosis as determined by TUNEL staining (0.48% vs. 0.05%, p < 0.01) and increased fibrosis as determined by Masson’s Trichrome staining (1.84% vs. 1.03%, p < 0.05) compared to Rb flox/flox mice after TAC.

In response to 4 weeks of TAC, cRb-KO mice showed increased heart weight (HW/TL: 12.93 ± 085 vs. 9.32 ± 0.34, p < 0.01), lung weight (LW) (LW/TL: 18.35 ± 2.66 vs. 10.21 ± 1.93, p < 0.01), cardiac dysfunction (EF: 34.5% ± 8.3 vs. 64.3% ± 8.9, p < 0.01), increased apoptosis as determined by TUNEL staining (0,42% vs. 0,18%, p < 0.05) and increased fibrosis as determined by Masson’s Trichrome staining (4.2 % vs. 1.1 %, p < 0.05) compared to Rb flox/flox mice after TAC.

Pressure gradients were similar between the cRb-KO mice submitted to 2 and 4 weeks of TAC and their respective controls.

In conclusion, our results suggest that endogenous Rb plays an important role in mediating cell survival in CMs and negatively regulates cardiac hypertrophy at baseline. Furthermore, we showed that the Rb protein is important for the maintenance of cardiac function in response to pressure overload.